Compositions, methods and microfluidics device for telomerase based in vitro diagnostic assays for detecting circulating tumor cells (ctc)

ABSTRACT

A repeatable method for detecting circulating tumor cells in vitro is provided. The method involves combining a test sample from a patient suspected of having circulating tumor cells, and a non-lytic adenoviral system, and culture media for the cells. The adenoviral system utilizes (i) a first replication-defective adenoviral particle in which an expression cassette is packaged, said expression cassette comprising an adenoviral 5′ and 3′ ITRs and a tumor-specific promoter; and (ii) a coding sequence for a reporter protein which is expressed in the presence of circulating tumor cells, and an adenoviral 3′ ITR. The test sample and the non-lytic adenoviral system are incubated for a sufficient time to permit expression of the reporter protein, and measuring reporter protein expression in the test samples, whereby presence of reporter expression indicates the presence of circulating tumor cells in the sample.

STATEMENT OF FEDERALLY SUPPORTED RESEARCH

This work was supported in part by the National Institutes of Health:NCI (RC1 CA145075) and NINDS (K08 NS076548-01). The US government mayhave rights in this invention.

BACKGROUND OF THE INVENTION

From their formation and throughout their development, primary tumorsshed cells that circulate through the bloodstream of cancer patients.These circulating tumor cells (CTCs) potentially hold important clinicalinformation that can be used for detection, characterization, accuratetreatment and monitoring of cancer. While the presence of CTCs in theblood has been documented for over 100 years and various methods havebeen described for their detection. [M. Alumni-Fabroni and M. Sandri,Methods, 50 (2010) 289-297], these traditional methods have lowsensitivity. Consequently, much of the research of the past decades hasbeen focused on the development of reliable methods for CTC enrichmentand identification, mainly trying overcome severe technical limitations.

In recent years, evidence supporting that the presence of CTCs in theblood can serve as a biomarker for potential disease and poor prognosishas continued to stimulate interest in the development of methodologiesfor detection and isolation of CTCs in blood samples from cancerpatients. Another important attribute that has made the detection andisolation of CTCs attractive is the fact that despite of theirheterogeneity, these cells may carry genetic information about theprimary tumor that can be useful in guiding the treatment of a specificpatient and providing an opportunity for individualize medicine.However, although many technologies have been recently developedrecently for the detection and isolation of CTCs from peripheral bloodsamples of cancer patients, this task remains technically challenging.

This is mainly due to the fact that CTCs are rare and only occur at verylow concentrations of one tumor cell per one billion blood cells.Typical methods for the identification and isolation of CTCs requiredextremely sensitive and specific analytical methods that generate verylow yield and purity samples.

Circulating tumor cell (CTC) assays may powerfully improve the abilityto monitor disease status, gauge prognosis, and guide treatmentdecisions for patients with cancer. However, CTC assays for manypatients including those with brain tumors (such as Glioblastomamultiforme (GBM)) have not been possible due to the lack of surfaceexpression of common biomarkers such as EpCAM to facilitate separationand subsequent detection. For other tumors such as non-small cell lungcancer (NSCLC), the ability to monitor treatment response may helpreduce the lethality of lung cancer by overcoming limitations of imagingto monitor NSCLC disease state intra and post treatment, and avoid theneed for a non-invasive means to assess NSCLC therapeutic effect andadjust the treatment plan accordingly.

The use of microfluidic devices for CTC detection and isolation has beendescribed in the literature. In general, a microfluidic device handlerelatively small cell numbers and sample volumes [e.g., from singlecells to millions of cells, and from 10 to 200 microliters] makingpossible the detection and isolation of CTCs from a sample which mayonly contain few CTCs. Currently, some microfluidic systems have beendescribed as useful for the detection of CTCs, including the CTC-Chip[S. Nagrath et “Isolating of rare circulating tumour cells in cancerpatients by microchip technology”, Nature, 450: 123:5-1239 (20 Dec.2007)], the herringbone-chip [S. L. Scott, et al, “Isolation ofcirculating tumor cells using a microvortex—generating herringbone chip,Proc Natl Acad Sci, vol. 107, no. 43, 18392-18397, e-pub Oct. 7, 2010,ahead of print Oct. 26, 2010] and the high-throughput microsampling unit(HTMSU) [A. Adams, et al, “Highly efficient circulating tumor cellisolation from whole blood and label-free enumeration usingpolymer-based microfluidics with an integrated conductivity sensor”, J.Am. Chem. Soc., 2008 Jul. 9; 130(27): 8633-41]. However, these systemspresent major technical limitations including the inability to capturenon-epithelial cells (e.g. cells which do not express epithelial celladhesion molecule (EpCAM). Capture efficiency and purity also meritimprovement (M. Alunni-Fabroni and M. T. Sandri, “Circulating tumorcells in clinical practice: Methods of detection and possiblecharacterization” Methods, 2010 Jan. 29; 50(4): 289-297).

Thus, there is still a need for a microfluidic device capable ofcapturing both epithelial and non-epithelial CTCs with high efficiencythat yields a CTCs sample with high purity for accurately characterizingthe biology of CTCs and to develop CTC analysis methodologies that canhelp guide diagnosis and treatment in a clinical setting. In addition,methods for qualitatively and/or quantitatively assessing CTC of avariety of tumor origins are still needed.

SUMMARY OF THE INVENTION

The invention provides a repeatable method for detecting circulatingtumor cells (CTC) in vitro. The method is repeatable because the cellsare not lysed as part of the assay and thus can remain viable in cultureto allow for secondary analysis.

In one aspect, the method involves combining a test sample from apatient suspected of having circulating tumor cells, a non-lyticadenoviral system, and culture media for the cells. The adenoviralsystem comprises: (i) a first replication-defective adenoviral particlehaving an adenoviral capsid in which an expression cassette is packaged,said expression cassette comprising an adenoviral 5′ ITR, antumor-specific promoter which is specifically activated in the presenceof circulating tumor cells, and an adenoviral 3′ ITR, wherein saidadenoviral particle is rendered replication defective and non-lytic: bya deletion in one or more adenoviral early genes; and (ii) a codingsequence for a reporter protein which is expressed in the presence ofcirculating tumor cells, and an adenoviral 3′ ITR. Following this, thetest sample and the non-lytic adenoviral system are incubated for asufficient time to permit expression of the reporter protein. Additionalculture media may be added or the culture media replenished. Reporterprotein expression is measured in the test samples whereby presence ofreporter expression indicates the presence of circulating tumor cells inthe sample. In one example, the promoter is human telomerase reversetranscriptase (hTERT). One or more of these steps may be repeated,optionally using a second adenoviral system with a different reporterprotein. The reporter protein may be under the control of the same or adifferent tumor specific promoter. Because the cells are not lysed inthis assay, the first and/or any subsequent measurement may be performedafter eighteen hours to ninety-six hours, or longer, after firstcombining the adenoviral system and the test sample.

In another embodiment, the invention provides a method for enriching atest sample from a patient suspected of having circulating tumor cells.The method involves obtaining a whole blood sample from a patient;combining the whole blood sample with neutral buffered saline in a tubehaving a gradient gel to separate out CTCs and white blood cells (WBCs)from red blood cells (RBCs), and a conical bottom; centrifuging the tubefor about 10 minutes to about 60 minutes at about 500 relativecentrifugal force (RCF) units to about 3000 RCF units; harvesting theCTC-enriched layer to obtain a CTC-enriched pellet suspension;incubating the pellet suspension in a water bath; and introducing cellculture media to allow pellet suspension in preparation for theadenoviral system.

In another aspect, the invention provides a product comprising anadenoviral vector system for use in a telomerase based assay fordetecting circulating tumor cells in vitro. The adenoviral system iscomposed of, at a minimum, (a) a first replication-defective adenoviralparticle having an adenoviral capsid in which an endonuclease expressioncassette is packaged, said expression cassette comprising an adenoviral5′ ITR, an hTERT promoter sequence which is activated in the presence ofcirculating tumor cells, an endonuclease coding sequence under thedirection of sequences which direct expression thereof, and anadenoviral 3′ ITR, wherein said adenoviral particle is renderedreplication defective by a deletion in one or more adenoviral earlygenes; and (b) a DNA molecule comprising an expression cassette for areporter gene comprising a coding sequence for the reporter protein andsequences which direct expression thereof, said expression cassetteflanked at its 5′ and 3′ ends by recognition sites for the endonuclease,and an adenoviral 3′ ITR.

In one embodiment, the reporter expression cassette (b) is on a secondreplication-defective adenoviral particle. The endonuclease can be arecombinase. In one embodiment, the reporter gene is selected from thegroup consisting of a green fluorescent protein, enhanced greenfluorescent protein, mCherry, red fluorescent protein, red fluorescentprotein turbo.

In still a further embodiment, the invention provides a microfluidicdevice for capturing circulating tumor cells (CTCs) from enrichedsamples. The devices is comprised of four capture channels containingdifferent arrays of microcolumns of different geometrical shapes andchemically functionalized with a biotin-binding protein that mediatesthe capture of biotinylated-antibody coated CTCs. The capture channelsare connected to an inlet and outlet reservoir through distributionchannels that facilitate the transport of sample in and out of thecapture channels. The arrangement of columns in this device is designedfor maximal capture, with greatest efficiency and purity (specificity)

These and other aspects of the invention will be readily apparent fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1F illustrates a CTC analysis performed according to theinvention. This illustrates the CTC-enrichment method (steps A to C)which include collection of whole blood (A), centrifuging in a tube witha gradient gel which separates CTC and white blood cells from red bloodcells (B), collection of the CTC-enriched layer and washing (C). Steps(D) (F) illustrate the CTC detection method of the invention, includingsuspending the CTC test sample in media and incubating prior to placingin the test vessel (D), adding the adenoviral vector system andincubating (E), detecting fluorescence (F) and optionally quantifyingusing a computer.

FIG. 2 is a schematic representation of a microfluidic device of theinvention. As illustrated, this device has four capture channelsarranged in parallel with an array of microcolumns with differentgeometrical shapes between channels. These channels are connected to aninlet and an outlet reservoir through a system of distribution channelsthat distribute facilitates the flow of sample into and out of thecapture channels.

FIG. 3A-FIG. 3I provide a schematic illustration of the double castingprototyping by thermal aging of PDMS process utilized for thefabrication of the PDMS cast used to make the microfluidic devicedescribed herein. The process is stated with a silica (Si) wafersubstrate (FIG. 3A). FIG. 3B illustrates Su-8 [a negative, epoxy-typephotoresist based on EPON SU-8 epoxy resin developed by and patent byIBM (U.S. Pat. No. 4,882,245—1989)] deposition and photolithography onthe Si wafer substrate. FIG. 3C illustrates the Su-8 mold. FIG. 3Dillustrates the use of the Su-8 mold for PDMS molding, curing andde-molding. FIG. 3E illustrates the thermal aging of the negative PDMSreplica of the Su-8 mold. FIG. 3F illustrates the resulting thermallyaged PDMS replica which serves as a mold to case a second PDMS replicawith positive orientation (FIG. 311). FIG. 3I illustrates the plasmaedging and bonding of the positive PDMS cast or replica of FIG. 3H to aglass slide.

FIG. 4A-FIG. 4C provides schematic diagrams of the device of FIG. 2.FIG. 4A provides a top view of the reservoir and distribution channelsystem. FIG. 4B provides a top view of the capture channels containedwithin the microfluidic device. FIG. 4C provides a cross-sectional viewof a capture channel of the device.

FIGS. 5A-5D provide enlarged views of the cylindrical microcolumns inone of the channels of the device of FIG. 2. FIG. 5A illustrates theshape and pattern of the cylindrical microcolumns. FIG. 5B is a furtherenlarged view of FIG. 5A and illustrates the size of one of thecylindrical shapes. FIG. 5C is an alternative further enlarged view ofFIG. 5A and illustrates a hexagonal-shaped unit of the cylindricalmicrocolumns. FIG. 5D is also an alternative further enlarged view ofFIG. 5A and illustrates the separation between the hexagonal units.

FIGS. 6A-6C provide enlarged views of the elliptical microcolumns in oneof the channels of the device of FIG. 2. FIG. 6A illustrates the shapeand pattern of the elliptical microcolumns. FIG. 6B is a furtherenlarged view of a single elliptical depression. FIG. 6C is an alternatefurther enlarged view of FIG. 6A and illustrates the separation betweenelliptical microcolumns from neighboring columns within a row andbetween rows.

FIGS. 7A-7D provides enlarged views of one of the microcolumns of thedevice of FIG. 2, which combines triangular and chevron-likemicrocolumns. FIG. 7A illustrates the shape and pattern of the trianglesand chevron-like microcolumn. FIG. 7B is a further enlarged view of FIG.7A showing the size and shape of a triangle. FIG. 7C is a furtherenlarged view of FIG. 7A showing the size and shape of a chevron. FIG.7D is a further enlarged view of FIG. 7A illustrating a triangle andchevron pattern, including orientation.

FIGS. 8A-8C, provides enlarged views of a second microcolumns of thedevice of FIG. 2 which combines triangular and chevron-likemicrocolumns. FIG. 8A the triangle and chevron pattern, which are in theopposite orientation to that of FIGS. 7A-D. FIG. 8B is a furtherenlarged view of FIG. SA showing the size between the twotriangle-chevron units and the space between the rows. FIG. 8B is afurther enlarged view of FIG. 7A showing the size and shape of thechevron. FIG. 8C is a further enlarged view of FIG. 8A illustrating thetotal size of a two triangle chevron unit, including the distancebetween the two triangle, and between the triangles and the chevron.

FIGS. 9A-9B are bright field and fluorescence images of U251 cells after4-8-hour incubation with 5×10⁹ GC/ml Ad.hTert.pCherry vector and beforebeing run through the microfluidic device. Most of the cells present inthe bright field image (FIG. 9A) also appear in the fluorescence image(FIG. 98) confirming that these cells are expressing the fluorescentfusion protein, pCherry. Fluorescence image was collected at 200 msec ofexposure time.

FIGS. 10A-10C illustrates in gray scale sections of tiled imagescollected for a representative capture channel. Channels are tiled inbright field and fluorescence using the red (FIG. 10B) and blue optic(FIG. 10C) channels, illustrated in gray scale. Bright field images(FIG. 10A) show the capture areas while the images collected withfluorescence channels show either captured cells that selectivelyexpress the fluorescent fusion protein, pCherry (infected U251 cells) inthe red channel (FIG. 10B) or all captured cells including ones notexpressing pCherry (non-infected U251 and Jurkat cells).

FIGS. 11A-11C are gray scale images in bright field (FIG. 11A) andfluorescence red (FIG. 11B) and blue (FIG. 11C) optic channels of cellscaptured at the surface of elliptical microcolumns in one of the capturechannels. FIG. 11A is a grayscale version of the bright field imagewhich illustrates that the captured cells are attached to differentsurfaces in the capture channel including the walls of the microcolumnsand the floor of the channel. FIG. 11B shows in grayscale that theimmobilized cells are positive for pCherry as shown in the fluorescenceimage taken with the red optics channel suggesting that they werecaptured via biatin-NeutrAvidin chemistry. In addition, fluorescenceimage taken with blue optics channel (FIG. 11C) confirms most of thecaptured cells are pCherry positive since all fluorescent points in thisimage dots to the fluorescent dots found in the red optics channelimage, thus suggesting high purity (in terms of infected cells only, notJurkats) of the captured cells.

FIG. 12 is a bar chart illustrating preliminary capture yield of themicrofluidic device described herein. Capture yield was calculatedindirectly by measuring the number of U251 cells positive for pCherryfound in the flow-through sample of individual experiments where either5.5×10³, 4×10⁴ or 8.5×10⁴ U251-infected cells (pCherry positive) mixedwith 1×10 Jurkat cells were injected into microfluidic devices.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting circulating tumorcells (CFCs) in vitro in a non-lytic system. Because the system isnon-lytic it offers additional flexibility in terms of timing of whenthe detection step may be performed and also offers additionalopportunity to verify and quantify CTCs, because the assay may berepeated on the same test sample because the cells of the test samplecan be maintained in culture. The system utilizes a non-lytic adenoviralvector system. Further, the invention provides a method for treating asample in order to enrich CTC and increase sensitivity and accuracy ofmeasurement. The enriched sample may thereafter be assessed usingconventional microscopy techniques. Alternatively, CTCs from theenriched sample may be assessed and enumerated using a microfluidicsdevice of the invention.

In one embodiment, the method for detecting CTC involves combining atest sample from a patient suspected of having circulating tumor cells,a non-lytic adenoviral system, and culture media for the cells.Suitably, the adenoviral system is composed of a replication-defectiveadenoviral particle having an adenoviral capsid in which an expressioncassette is packaged. The adenoviral vector contains an adenoviral 5′ITR, a tumor-specific promoter which is specifically activated in thepresence of circulating tumor cells, and an adenoviral 3′ ITR. Theadenoviral particle is rendered replication defective and non-lytic,e.g., by a deletion in one or more adenoviral early genes. Theadenoviral vector system further comprises a coding sequence for areporter protein which is specifically expressed in the presence ofcirculating tumor cells, and an adenoviral 3′ ITR. The test sample andthe non-lytic adenoviral system are incubated for a sufficient time topermit expression of the reporter protein, during which time the cellsare maintained by replenishment or addition of fresh media. Followingincubation, reporter protein expression is measured in the test samples.Since expression of the reporter gene is under the specific control of atumor associated promoter or a telomerase promoter, reporter geneexpression indicates tumor cells in the sample. These steps mayoptionally be repeated.

Non-Lytic Adenoviral Vector Systems:

The method of the invention utilizes an adenoviral vector system inwhich the adenoviral vector is rendered replication defective in amanner which prevents its ability to lyse the target cells while stillpermitting efficient infection thereof. Several variants of theadenoviral vector system are provided by the present invention. In oneembodiment, the assay utilizes a single adenoviral vector. However, evenin embodiments which utilize two or more, i.e., multiple,replication-defective adenoviral vectors, each of the adenoviral vectorsis permits the cells to be infected and the assay to proceed to CTCdetection without cell lysis.

The adenoviral vector system utilizes one or more adenoviral particle,which particle has an adenoviral capsid into which is packaged selectedadenoviral nucleic acid sequences which have had the regions responsiblefor replication function ablated, either by deletion of adenoviralcoding regions, or by insertion and disruption of the coding regions. Inone embodiment, the adenoviral particles are renderedreplication-defective by deletions in one, or preferably both, the E1aand/or E1b genes. Alternatively, the adenoviruses are renderedreplication-defective by another means, optionally while retaining theE1a gene or E1b gene (e.g., an insertion which disrupts expression ofthe E1a and/or E1b gene product). Similarly, in some embodiments,reduction of an rune response to the vector may be accomplished bydeletions in the E2b and/or DNA polymerase genes. The adenoviral vectorscan also contain other mutations to the adenoviral genome, e.g.,temperature-sensitive mutations or deletions in other genes.

The adenoviral vector systems are designed so that a reporter isexpressed in the presence of circulating tumor cells, but not in theabsence of such CTCs. In order to accomplish this, the adenoviral vectorsystem is designed so that the reporter is expressed under the controlof a promoter which is specifically activated in the present ofcirculating tumor cells. In one embodiment, the promoter is a telomeraseassociated promoter, such as human telomerase reverse transcriptase(hTERT) [Lim, K. W., et al, Coexistence of two distinct G-quadruplexconformations in the hTERT promoter, J. Am. Chem. Soc. 132 (35),12331-12342 (2010): U.S. Pat. No. 6,610,839], a human telomerase RNA(hTR), a hTERC promoter [Glasspool, R M, et al, “The hTERT and hTERCTelomerase Gene Promoters Are Activated by the Second Exon of theAdenoviral Protein, E1A, identifying the Transcriptional CorepressorCtBP as a Potential Repressor of Both Genes”, Neoplasia. 2005 June;7(6): 614-622], and a progression elevated gene promoter (PEG-Prom)[See, e.g., US20040203066A1, for the sequences of the rat PEG-Prompromoter; Su Z Z, et al., Proc Natl Acad Sci USA. 2005 Jan. 25;102(4):1059-64. Epub 2005 Jan. 12. “Targeting gene expressionselectively in cancer cells by using the progression-elevated gene-3promoter”]. For convenience in this specification, reference will bemade to a telomerase-specific promoter. However, it will be understoodthat tumor specific promoters may also be utilized in the constructs andmethods described herein.

Suitably, this promoter is operably linked to the sequence encoding areporter protein, in one embodiment, the reporter protein is afluorescent protein. A variety of fluorescent proteins have beendescribed in the literature. These proteins and their coding sequencesare available from a variety of sources including commercial sourcessuch as, e.g., BioVision, EMD Invitrogen, amongst other sources.Suitable proteins include, green fluorescent protein, enhanced greenfluorescent protein, mCherry, red fluorescent protein, and redfluorescent protein turbo, amongst others. However, other suitableproteins may be selected.

Fluorescent Proteins and Properties Thereof Relative Excitation EmissionMolar Brightness Protein Maximum Maximum Extinction Quantum in vivo (%of (Acronym) (nm) (nm) Coefficient Yieid Structure EGFP) GFP (wt)395/475 509 21,000 0.77 Monomer* 48 Green Fluorescent Proteins EGFP 484507 56,000 0.60 Monomer* 100 Emerald 487 509 57,500 0.68 Monomer* 116Superfolder 485 510 83,300 0.65 Monomer* 160 GFP Azami 492 505 55,0000.74 Monomer 121 Green mWasabi 493 509 70,000 0.80 Monomer 167 TagGFP482 505 58,200 0.59 Monomer* 110 TurboGFP 482 502 70,000 0.53 Dimer 102AcGFP 480 505 50,000 0.55 Monomer* 82 ZsGreen 493 505 43,000 0.91Tetramer 117 T-Sapphire 399 511 44,000 0.60 Monomer* 79 Blue FluorescentProteins EBFP 383 445 29,000 0.31 Monomer* 27 EBFP2 383 448 32,000 0.56Monomer* 53 Azurite 384 450 26,200 0.55 Monomer* 43 mTagBFP 399 45652,000 0.63 Monomer 98 Cyan Fluorescent Proteins ECFP 439 476 32,5000.40 Monomer* 39 mECFP 433 475 32,500 0.40 Monomer 39 Cerulean 433 47543,000 0.62 Monomer* 79 mTurquoise 434 474 30,000 0.84 Monomer* 75 CyPet435 477 35,000 0.51 Monomer* 53 AmCyan1 458 489 44,000 0.24 Tetramer 31Midori-Ishi 472 495 27,300 0.90 Dimer 73 Cyan TagCFP 458 480 37,000 0.57Monomer 63 mTFP1 462 492 64,000 0.85 Monomer 162 (Teal) YellowFluorescent Proteins EYFP 514 527 83,400 0.61 Monomer* 151 Topaz 514 52794,500 0.60 Monomer* 169 Venus 515 528 92,200 0.57 Monomer* 156 mCitrine516 529 77,000 0.76 Monomer 174 YPet 517 530 104,000 0.77 Monomer* 238TagYFP 508 524 64,000 0.60 Monomer 118 PhiYFP 525 537 124,000 0.39Monomer* 144 ZsYellow1 529 539 20,200 0.42 Tetramer 25 mBanana 540 5536,000 0.7 Monomer 13 Orange Fluorescent Proteins Kusabira 548 559 51,6000.60 Monomer 92 Orange Kusabira 551 565 63,800 0.62 Monomer 118 Orange2mOrange 548 562 71,000 0.69 Monomer 146 mOrange2 549 565 58,000 0.60Monomer 104 dTomato 554 581 89,000 0.69 Dimer 142 dTomato- 554 581138,000 0.69 Monomer 283 Tandem TagRFP 555 584 100,000 0.48 Monomer 142TagRFP-T 555 584 81,000 0.41 Monomer 99 DsRed 558 583 75,000 0.79Tetramer 176 DsRed2 563 582 43,800 0.55 Tetramer 72 DsRed- 555 58438,000 0.51 Tetramer 58 Express (T1) DsRed- 556 586 35,000 0.10 Monomer10 Monomer mTangerine 568 585 38,000 0.30 Monomer 34 Red FluorescentProteins mRuby 558 605 112,000 0.35 Monomer 117 mApple 568 592 75,0000.49 Monomer 109 mStrawberry 574 596 90,000 0.29 Monomer 78 AsRed2 576592 56,200 0.05 Tetramer 8 mRFP1 584 607 50,000 0.25 Monomer 37 JRed 584610 44,000 0.20 Dimer 26 mCherry 587 610 72,000 0.22 Monomer 47 HcRed1588 618 20,000 0.015 Dimer 1 mRaspherry 598 625 86,000 0.15 Monomer 38dKeima- 440 620 28,800 0.24 Monomer 21 Tandem HcRed- 590 637 160,0000.04 Monomer 19 Tandem mPlum 590 649 41,000 0.10 Monomer 12 AQ143 595655 90,000 0.04 Tetramer 11 *Weak Dimer

Where the assay is performed multiple times with different repot tergenes, or where a multi-adenoviral system is utilized which alsoutilizes two or more different reporter genes, it is desirable that theproteins used are readily distinguishable from one another. For example,one may select a green fluorescent protein and a red fluorescentprotein; or a green fluorescent protein and a yellow fluorescentprotein, or a tangerine fluorescent protein and a yellow fluorescenceprotein. Still other suitable combinations may be selected.

In yet another embodiment, the vector system expresses a fusion proteinwhich comprises a protein, polypeptide or peptide fused in frame to afluorescent protein or a fragment thereof which functions (fluoresces)in the cell fused. In one embodiment, the fusion partner for thefluorescent protein or functional fragment thereof is an epithelial celladhesion molecule such as EpCAM, or a functional fragment thereof. Suchfusion proteins may be constructed using techniques such as described inC. Y. Wang, et al, 2004 November; 20(6): 765-768; K. Slanchev et al,PLoS Genet 2009.

The vector system provides a sequence encoding a reporter which isexpressed directly or indirectly under the control of atelomerase-specific promoter. The expression cassette with the reportercoding sequence also may contain other regulatory control sequencesnecessary for expression of the reporter protein, including, e.g., anenhancer, a polyA, amongst other elements.

The reporter sequence may be located in the site of one of the deletedadenovirus early genes, e.g., E1a, E1b, E2a, E3, or E4a. Optionally, anadenovirus may contain two more reporter gene expression cassetteslocated either in tandem or in different adenovirus early gene sites(e.g., both in the region and in E3) of a single adenovirus vector.

In another embodiment, a single adenovirus contains two differentexpression cassettes located in different early gene sites. In thisembodiment, a first expression cassette contains the inducing orregulating agent under the control of a telomerase specific promoter anda second expression cassette contains a reporter gene under the controlof an inducible or regulatable system. In this embodiment, theexpression cassette with the inducing agent to be located in the site ofthe adenovirus E1 deletion and the expression cassette with thefluoroprotein coding sequence to be in the site of the adenovirus E3deletion. However, other arrangements in the adenovirus may beengineered by one of skill in the art utilizing known geneticengineering and recombinant techniques. In one embodiment, a singleadenovirus system with the dual cassette system can provide selfamplifying effects. The principle of this vector system is that whenTelomerase activity is present in a transduced cell (such as a CTC), thepromoter (e.g., hTERT) is activated and drives expression of thenuclease. The nuclease then causes inversion of a previously invertedfluorescent protein cassette (flanked by the two nuclease recognitionsites), inducing expression of the Fluor-Protein. The inversion of theFluor-Protein cassette then allows expression of the Fluor-Protein andconsequently the targeted CTC fluoresces and is detectable.

In one embodiment, the fluoro-protein cassette is under the control of aconstitutive promoter which drives its expression. In one embodiment,the promoter is a strong constitutive promoter, e.g., the CAGESpromoter, which composed of the chicken β-actin promoter with humancytomegalovirus immediate early (CMVIE) enhancer.

In still another embodiment, this two expression cassette system may becarried by two different adenoviruses, one which carries the expressioncassette with the reporter gene in an inducible or regulatable systemand a second adenovirus which carries the inducing or regulating agentwhich is expressed by a telomerase specific promoter in the presence oftumor cells. Optionally, each of the adenoviruses contains two or morecopies of the reporter gene or the inducing/regulating agent,respectively. When utilizing a multiple vector system, the twoexpression cassettes may each be located within the E1 region of theirrespective adenoviruses. Alternatively, the site of the E3 deletion maybe selected for insertion of the expression cassette in one or more thanone of the adenoviruses.

As described herein, the reporter protein may be controlled by anexpression control system. In one embodiment, the expression cassettefor the marker gene contains an endonuclease recognition site locatedboth 5′ and 3′ to the coding sequence for the reporter protein and itsupstream expression control sequences (e.g., promoter, any enhancer,intron) and the other expression cassette in the system contains anendonuclease coding sequence under the control of the telomerasespecific promoter. The regulatable system can be selected from atet-on/tet-off system, a tetR-KRAB system, a mifepristone (RU486)regulatable system, a tamoxifen-dependent regulatable system, arapamycin-regulatable system, or an ecdysone-based regulatable system.In one embodiment, the system utilizes an endonuclease, a recombinase, ameganuclease, or a zinc finger endonuclease that binds to the ablationrecognition site in the first transcription unit and excises or ablatesDNA and an interfering RNA, a ribozyme, or an antisense that ablates theRNA transcript of the reporter expression cassette. In one embodiment,the endonuclease is a Cre recombinase and the recognition sites flankingthe reporter transgene are loxP sites. In another embodiment,endonuclease is FLY and the recognition site is FRT. In anotherembodiment, the endonuclease is a chimeric endonuclease such as thatdescribed in WO 2011/126808 A2. Another suitable endonuclease may beselected from among intron endonucleases, such as, e.g., I-T evil. Stillother suitable nucleases include, e.g., integrases (catalyze integrationserine recombinases (catalyze recombination), tyrosine recombinases,invertases (e.g. Gin) (catalyze inversion), resolvases, Tn3), andnucleases that catalyze translocation, resolution, insertion, deletion,degradation or exchange. Such nucleases have been described in theliterature and are available from a variety of commercial sources.

In another embodiment, the reporter protein is expressed under thecontrol of a constitutive promoter, e.g., when a dual or multi-vectorsystem is utilized. Examples of constitutive promoters include, withoutlimitation, the TBG promoter, the retroviral Rous sarcoma virus (RSV)LTR promoter (optionally with the RSV enhancer), the cytomegalovirus(CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart etal, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter [Invitrogen]. A number of otherexpression control sequences, including promoters which are nativeand/or tissue-specific, are known in the all and may be utilized.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signalsincluding rabbit beta-globin polyp; sequences that stabilize cytoplasmicmRNA; sequences that enhance translation efficiency (e.g., Kozakconsensus sequence); sequences that enhance protein stability; and whendesired, sequences that enhance secretion of the encoded product. Amongother sequences, chimeric introns may be used.

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Onecan enhance expression of desired proteins by known means to enhance theeffectiveness of this system. For example, using the Woodchuck HepatitisVirus Posttranscriptional Regulatory Element (WPRE). In a furtherembodiment, other expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In a vector system of the invention which utilizes two or more non-lyticadenoviruses, the vector elements of the two adenoviruses may beindependently selected. The use of two different reporter genes has alsobeen described for use in sequential assay steps. These two differentreporter genes may be under the control of the same or different tumorspecific promoters and/or the same or different nucleases. This may alsobe true of a single adeno viral vector containing two different reportergenes within a single adeno viral particle. In such an instance, thereporter genes are distinguishable from one another. Further, theinvention contemplates the use of two or more non-lytic, adenoviruses,whether used sequentially or simultaneously, in which two differentreporter genes are under control of two different promoters. Forexample, a first reporter gene may be expressed in the presence of atelomerase-specific promoter and a second reporter gene may be selectedwhich is expressed only in the presence of a specific type of tumor.Still other variations will be apparent to one of skill in the art inview of the information provided herein.

Production of Replication-Defective Adenoviruses Replication-defectiveadenoviral vectors may be produced using methods which are known in theart, e.g., through use of adenoviral plasmids. In one embodiment, theadenoviral vectors are adenoviral particles which are renderedreplication-defective by deletions in the E1a and/or E1b genes. Theparticle is composed of an adenoviral capsid which directs targeting ofthe adenoviral vector. Because this assay is performed in vitro,limitations on in vivo use of adenoviruses based on pre-existingimmunity in the patient population to the adenovirus capsid (andparticularly the capsid) are not a factor in the selection of theadenovirus source for the non-lytic vectors described herein. Rather,adenoviruses are selected taking into consideration such factors as easeof production and ability to target and infect cells efficiently. In theexamples described herein, is adenovirus is human adenovirus 5 [VR-5,American Type Culture Collection]. However, an adenovirus from anothersource, particularly those naturally or modified to have particularaffinity for circulating tumor cells in mammals, including humans, maybe readily selected. Such an adenovirus may be of human origin,including, without limitation, Ad2, Ad31, Ad36 and Ad37. In otherembodiments, the adenovirus may be of simian or another animal origin.Examples of suitable simian adenoviruses include those described in,e.g., U.S. Pat. No. 8,105,574 (Pan5, Pan6, Pan7, SV1, SV25, SV39), WO2012/071318 (A1321, A1325, A1295, A1316, A1322), WO 2009/073104; WO2009/105084; and WO 2009/073102. Still other adenoviruses are known andmay be obtained from a variety of sources including the ATCC, commercialand academic sources, or the sequences of the Ad may be obtained fromGenBank or other suitable sources. The adenoviral vectors can alsocontain other mutations to the adenoviral genome, e.g.,temperature-sensitive mutations or deletions in other genes. In otherembodiments, it is desirable to retain an intact E1a and/or E1b regionin the adenoviral vectors. Such an intact E1 region may be located inits native location in the adenoviral genome or placed in the site of adeletion in the native adenoviral genome (e.g., in the E3 region).

Thus, one embodiment, the system incorporates a vector in which theviral sequences contain mutation that renders it temperature sensitive.At the lower “permissive” temperature of 32° Celsius; the virus is ableto replicate within transduced cells and thus amplify the fluorescentsignal. However, before the replication process can proceed to celllysis (and thus death of the transduced CTC), the cells are shifted tothe higher “non-permissive” temperature of 37° C. At that highertemperature, all replication ceases and the CTCs should stay intact.This system has the potential weakness however of “leakiness”, in whicha degree of viral replication may still occur at the higher temperature,resulting in CTC lysis. There is also the technical difficulty that theimage acquisition and analysis and subsequent processing of CTCs mayunavoidably involve conditions that make maintaining the highernon-permissive temperature not possible.

A range of adenovirus nucleic acid sequences can be employed in thevectors. For example, all or a portion of the adenovirus delayed earlygene E3 may be eliminated from the adenovirus sequence which forms apart of the recombinant virus. The function of an E3 is believed to beirrelevant to the function and production of the recombinant virusparticle. Adenovirus vectors may also be constructed having a deletionof at least the ORF6 region of the E4 gene, and more desirably becauseof the redundancy in the function of this region, the entire E4 region.Still another vector of this invention contains a deletion in thedelayed early gene Eta. Deletions may also be made in any of the lategenes L1 through L5 of the adenovirus genome. Similarly, deletions inthe intermediate genes IX and IVa₂ may be useful for some purposes.Other deletions may be made in the other structural or non-structuraladenovirus genes. The above discussed deletions may be usedindividually, i.e., an adenovirus sequence for use as described hereinmay contain deletions in only a single region. Alternatively, deletionsof entire genes or portions thereof effective to destroy theirbiological activity may be used in any combination. For example, in oneexemplary vector, the adenovirus sequence may have deletions of the E1genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 andE3 genes, or of E1, E2a and E4 genes, with or without deletion of E3,and so on. As discussed above, such deletions may be used in combinationwith other mutations, such as temperature-sensitive mutations, toachieve a desired result.

An adenoviral vector lacking any essential adenoviral sequences (e.g.,E1a, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured in thepresence of the missing adenoviral gene products which are required forviral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)or a packaging host cell. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication WO96/13597, published May 9, 1996, and incorporated hereinby reference.

Helper Viruses

Thus, depending upon the adenovirus gene content of the viral vectorsemployed to carry the expression cassette, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientadenovirus gene sequences necessary to produce an infective recombinantviral particle containing the expression cassette. Useful helper virusescontain selected adenovirus gene sequences not present in the adenovirusvector construct and/or not expressed by the packaging cell line inwhich the vector is transfected. In one embodiment, the helper virus isreplication-defective and contains a variety of adenovirus genes inaddition to the sequences described above. Such a helper virus isdesirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 374:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus may optionally contain a second reporter minigene. A number ofsuch reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the transgene on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

2. Complementation Cell Lines

To generate recombinant adenoviruses (Ad) deleted in any of the genesdescribed above, the function of the deleted gene region, if essentialto the replication and infectivity of the virus, must be supplied to therecombinant virus by a helper virus or cell line, i.e., acomplementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the Advector. This is particularly advantageous because, due to the diversitybetween the Ad sequences of the invention and the human AdE1 sequencesfound in currently available packaging cells, the use of the currenthuman E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line which expresses the E1 gene products that can beutilized for production of an E1-deleted adenovirus, Such cell lineshave been described. See, e.g., U.S. Pat. No. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from the same adenovirus as supplies the adenoviral vectorcapsid or a transcomplementary adenovirus under the transcriptionalcontrol of a promoter for expression in a selected parent cell line.Inducible or constitutive promoters may be employed for this purpose.Examples of such promoters are described in detail elsewhere in thisspecification. A parent cell is selected for the generation of a novelcell line expressing any desired adenoviral gene. Without limitation,such a parent cell line may be HeLa [ATCC Accession No. CCL 2], A549[ATCC Accession No. CCL 185], HEK 293, KB [CCL 17], Detroit [e.g.,Detroit 510, CCL 72] and WI-38 [CCL 75] cells, among others. These celllines are all available from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209. Other suitable parentcell lines may be obtained from other sources.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3,10T1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, F J et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

3. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering a vector for production of the adenoviralparticle, the adenoviral vector is delivered in an amount from about 5μg to about 100 μg DNA, and preferably about 10 to about 50 μg DNA toabout 1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

The packaging or production vector may be any vector known in the art ordisclosed above, including naked DNA, a plasmid, phage, transposon,cosmids, episomes, viruses, etc. Introduction into the host cell of thevector may be achieved by any means known in the art or as disclosedabove, including transfection, and infection. One or more of theadenoviral genes may be stably integrated into the genome of the hostcell, stably expressed as episomes, or expressed transiently. The geneproducts may all be expressed transiently, on an episome or stablyintegrated, or some of the gene products may be expressed stably whileothers are expressed transiently. Furthermore, the promoters for each ofthe adenoviral genes may be selected independently from a constitutivepromoter, an inducible promoter or a native adenoviral promoter. Thepromoters may be regulated by a specific physiological state of theorganism or cell (i.e., by the differentiation state or in replicatingor quiescent cells) or by exogenously-added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaguing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredexpression cassette-containing viral vector, the vector is transfectedin vitro in the presence of a helper virus into the packaging cell line,Homologous recombination occurs between the helper and the vectorsequences, which permits the adenovirus-transgene sequences in thevector to be replicated and packaged into virion capsids, resulting inthe recombinant viral vector particles. However, the invention is notlimited to the method for production of the adenoviral vectors.

The resulting replication-defective adenoviruses are useful in themethods of the system and assay of invention.

Circulating Tumor Cell (CTC) Test Sample Preparation and CTC DetectionAssay:

The invention utilizes one or more adenoviruses which have been renderednon-lytic and which specifically express a fluorescent protein in thepresence of circulating tumor cells to qualitatively and quantitativelydetect the presence of CTCs. As described above, the method fordetecting circulating tumor cells in vitro involves combining a testsample from a patient suspected of having circulating tumor cells and anon-lytic adenoviral system in a well, tube or another vessel containingmedia sufficient to permit infection of the cells and sustain the cellsthroughout the assay period. The test sample and the non-lyticadenoviral system are incubated under conditions which permit cellinfection and which permit expression of the reporter protein. The cellmedia may be replaced or replenished as needed throughout this process.After the desired incubation period, reporter protein expression ismeasured in the test samples. As described herein, because the system isnon-lytic, the steps described herein, including infection, incubationand/or measurement steps may be optionally repeated, either with thesame or a different non-lytic reporter adenovirus system.

CTC Enrichment

In one embodiment, a whole blood sample is taken from a subject andenriched prior to being used as a test sample in the method describedherein. This is done because of the relatively low number of circulatingtumor cells in the bloodstream and to improve the accuracy of results.Various techniques and apparatus fir enriching samples with circulatingtumor cells have been described in the literature, including speciallyengineered parlene membrane microfilters [Lin L K, et al, Clin CancerRes, 2010, 6(20):5011-5018], a density gradient separation method whichis commercially available as OncoQuick® tubes [Greiner Bio-One,Frickenhausen, Germany], a CellSearch system (Veridex LLC), which relieson ferrofluid particles coated with epithelial cell adhesion molecule(EpCAM) antibodies to magnetically extract CTCs [Allard W J, at al, ClinCancer Res, 2004 Oct. 15; 10(20): 6897-6904], a circulating tumor cellmicrochip, which utilizes anti-EpCAM antibodies bound to a silicon-basedmicrochip over which blood samples are passed [Nagrath L V et al, Nature2007; 450 (7173): 1235-1239]. Still other cell enrichment and extractionmethods are described by Mikolajezyk et at [Mikolajczyk D S, et al, JOncol 2011; 2011: 252361]. However, while these methods can be used inconjunction with the CTC detection assay of the method, none of thesemethods are as optimal of the CTC enrichment method described in thepresent application.

For the CTC enrichment of the present invention, a whole blood sample isobtained from a patient. Typically, between about 1 mL to about 15 mL,or about 5 mL to 12 mL, or about 10 mL is drawn. The blood sample thiscombined with a neutral buffered saline. For example, phosphate bufferedsaline may be selected. Alternatively, another buffered saline may beused. In one embodiment, approximately equivalent amounts of the bloodand buffered saline are combined. However, variations on thiscombination may be selected, e.g., where more blood or more bufferedsaline are used. The blood-saline mixture are optionally typicallychilled to about 0° C. to about 4° C. prior to centrifuging. Theblood-saline mixture is placed in a test tube with a conically shapedbottom, which tube contains a density gradient separation fluid and arigid but porous disk that minimizes mixing of blood with the gradientseparation medium while the blood mixture is being added to the tube. Asuitable system is commercially available from OncoQuick®, However,other separation systems could be substituted therefor. The tube is thencentrifuged to separate the CTC from peripheral blood cells withoutlysing the cells, including, e.g., hematopoietic cells, erythrocytes,and granulocytes. In one embodiment, the tube is centrifuged for about10 minutes to about 60 minutes at about 500 relative centrifugal force(REF) units to about 3000 RCF units. In one embodiment, centrifuging isperformed for about 20 minutes at about 1500 RCF units. However, thetimes and RCF units described herein can be varied to achieve cellseparation while avoiding cell lysis or disruption. The enriched CTCslayer is harvested, typically in the form of a CTC-enriched pelletsuspension. The CTC-enriched pellet is resuspended, and may be incubatedin a water bath into which cell culture media is introduced to allowpellet suspension in preparation for the adenoviral system. Such a waterbath is typically at about body temperature (e.g., about 35 CC to about40° C., more preferably about 37° C.), although variations arepermitted. In certain embodiments, following collection of the firstCTC-enriched pellet suspension, the remaining solution is extracted andplaced in a separate vessel, and the original vessel is washed in orderto recapture any residual CTCs which are subsequently processed asdescribed for the other CTCs and combined with the first CTC-enrichedpellet suspension for inclusion in the test sample.

The enriched CTC test sample and culture media are aliquotted intochamber wells, a petri dish, tube, or another suitable vessel forconducting the CTC detection assay of the present invention, Whetherusing a single non-lytic adenovirus system or a multiple non-lyticadenovirus system as used herein, the total of adenoviruses combinedwith the test sample is about 10⁵ to about 10¹⁴ adenoviral genomes (GC)or viral particles (VP), about 10⁷ to about 10¹² GC or VP, or about 10⁸to about 10¹⁰ GC or VP, per 50 μL. Each well may have about 1000 toabout 1,000,000 (total) cells; this typically includes both white bloodcells and CTCs. In one embodiment, adenovirus vectors are introduced ata concentration of 2×10⁸ viral particles/50 The adenoviral vectors maybe combined together and added simultaneously to the cells, or combinedseparately into the cells; such that the combined amount of adenovirusesadded to the cells is within ranges set forth above. The ratio of twodifferent adenoviral vectors may be about 2:about 1 to about 1:about 2VP, or this ratio may be varied. For example, in one embodiment, twodifferent adenoviral vectors are added in a ratio of about 1 to about 1,In another embodiment, e.g., where the system is self-amplifying, it maybe desirable to have a ratio of about 2:about 1, wherein the amount ofvector containing the nuclease is present in an amount of about 2 timesthe amount of the adenovirus with the marker gene.

The test sample is then incubated under conditions suitable to permitcontinued cell viability. In one embodiment; the cells are incubated forabout eighteen hours to ninety-six hours, or about twenty-four hours tothirty-six hours, before measuring reporter expression levels. However;shorter or longer incubation times may be selected. For example, whenusing a single adenoviral vector system which does not self-amplify, itmay be desirable to use longer incubation times and to select proteinswhich have high signal to noise ratios (e.g., bright fluorescenceagainst low background autofluorescence).

Optionally, incubation is at about 3° C. in a humidified chamber under5% CO₂, but other suitable conditions may be selected after firstcombining the adenoviral system and the test sample. Followingincubation, expression of the report may be performed using methods suchas are known in the art including, e.g., a fluorescent microscopyimaging. The assay may be repeated a second time, or more, using thesame, or a different non-lytic adenoviral system of the invention,optionally with a different fluorescent protein than in the firstnon-lytic adenoviral system. Following imaging, the reaction vessel(e.g., chamber wells) can be fixed and counterstained to qualitativelyassess isolated circulating tumor cells. The shape, size, and appearanceof each fluorescent object helps confirm that individual CTCs aredetected, and so that debris, parts of cells, or clumps of cells can beexcluded from the analysis. A number of computer programs and algorithmsare available which assist in enumeration by established parametersincluding fluorescent intensity, cell area, and cell diameter, such asIP Lab (Scanalytics (Spectra Services)).

The imaging described above can show both white blood cells (WBCs) andCTCs, e.g. on light (phase) microscopy, or under fluorescent microscopyif the nuclei of both WBCs and CTCs are stained with a nuclear markersuch as Hoechst or DAN dye. However, only the CTCs will showfluorescence, such as GFP, due to the telomerase activity driving thevector system. In contrast, the WBC will not be fluorescent or show onlybackground fluorescence. The number of cells that express GFP underthese conditions can then be counted and calculated as the number ofCTCs per ml of original blood from the patient at the beginning of theassay, Secondary staining may also be performed, to further confirm thenature of the CTCs. For example, the purified cells can be stained forEpCAM (e.g. for lung cancer) or Nestin (for Glioblastoma Multiforme((IBM)). WBCs would not stain for EpCAM and Nestin, while CTCs wouldshow both GFP and the stain for the secondary marker (which in turn isdetected with secondary antibodies conjugated to a differentfluorochrome, such as RFP). Alternatively, the WBCs can be stained for aWBC marker such as CD45, which would not be expressed by the CTC.Consequently, a typical result could consist of GFP-expressing CTCs donot show CD45, in the midst of many CD45-expressing WBCs that do notshow GFP.

In a further embodiment, the invention provides a product comprising anovel vector system for use in a telomerase based assay for detectingcirculating tumor cells in vitro. The product may contain one or morenon-lytic adenoviral particles, optionally in freeze-dried state; asuspending agent; tubes, pouches, vials, syringes, or other suitablecomponents of a kit.

Microfluidic Device

In one aspect, the invention provides a novel circulating tumor cell(CTC-specific) isolation device that enables the identification andcapture of viable CTCs for detection, propagation and molecularcharacterization purposes. Although not so limited, the presentinvention is particularly well suited for identification and capture ofviable CTCs derived from peripheral blood samples. The inventionprovides for the selected tagging of enriched CTCs with anextracellular, detectable (e.g., fluorescent) epitope via adenoviraltransduction and the use of easy-release, biotinylated primaryantibodies against this epitope to mediate CTC immunocapture andisolation by means of a biotin-binding protein coated microfluidicdevice. Cancer cell-specific tagging is achieved as described herein,e.g., by exposing an enriched CTC sample, prepared by liquid densityseparation of a whole blood sample, to an adenovirus that delivers aspecific episomal marker.

The selectivity of the CTC tagging is conferred by the use of theadenoviral vector system described in this specification. However,alternative systems may be used with the device of the invention. Forexample, a fusion protein composed of epithelial cell adhesion molecule(EpCAM) and green fluorescent protein (GFP) may be delivered via anadenoviral vector for capture in the CTC microfluidic device of theinvention.

Existing (prior art) methods for capture and/or detection of CTCs dependon the endogenous expression of specific proteins typically associatedwith epithelial cells. These proteins include EpCAM and cytokeratins(Ck). However, consideration differences in expression profile of theseproteins may exist between CTC samples or even among tumor cells in asingle sample. This limits the population of CTCs that can be capturedand/or detected by these methods to only those cells expressing thesespecific epithelial cell proteins. Furthermore, due to the process knownas Epithelial-Mesenchymal-Transition (EMT), EpCAM expression may be downregulated or absent in CTCs. Without wishing to be bound by theory, itis believed that by selectively tagging CFCs via an adenoviral deliveredexogenous epitope as described above, the method and device of theinvention extends the phenotypic range of CTC populations that can becaptured and/or detected and simplifies the process of immunocapture andidentification of these cells. Immunocapture of tagged CTCs isaccomplished by incubating cells with easy-release, biotinylatedantibodies against the lagging epitope, then by flowing them through amicrofluidic device of the invention containing biotin-binding protein(NeutraAviden™ deglycosylated avidin protein) coated microcolumns. Theincubation step with the early-release, biotinylated antibody allows thecell surface of CTCs to be bound with biotin molecules that are used tocapture these cells with the microfluidic device through biotin-avidinchemistry. The microfluidic device of the invention is described below.The device contains four capture channels, each containing an array ofmicrocolumns of various shapes that include cylindrical, oval and acombination of triangular and chevron-like structures arranged indifferent orientations.

In one embodiment, easy-release, biotinylated antibodies are used tomediate the capture of CFCs, including the EL-Link® NHS-SS-PEG4-Biotinlabeling system (Pierce, Thermo Scientific) and the DSB-X™ Biotinlabeling system (Molecular Probes, Invitrogen). The EZ-Link®NHS-SS-PEG4-Biotin labeling system allows labeled antibodies to becaptured with biotin molecules containing a flexible spacer arm with areducible disulfide bond. The DSB-X™ Biotin labeling system allows aprecursor of biotin, desthiobiotin, to be incorporated. This precursorbinds biotin-binding proteins and can be readily displaced by excessD-biotin. Still other suitable easy-release biotinylated antibodies maybe selected or designed.

Without wishing to be bound by theory, this easy-release system isbelieved to be advantageous over the prior art methods because the priorart chemical and physical methods required to overcome the strongbinding forces used to immobilize CTCs onto micro-columns of amicrofluidic device are known in the art to adversely affect cellviability and function.

Once captured and visualized (e.g., by observing fluorescence), CTCs areimaged, identified and enumerated in a transparent PDMS microfluidicdevice using a suitable detection system, e.g., a sensitive fluorescentmicroscope (Eclipse TE2000-U, Nikon Corp), and automated stage and animage analysis program (e.g., Image-Pro Plus 7.0, Media Cybernetics,Rockville, Md.).

CTCs are immunocaptured within the microfluidic device. Immunocapture ofCFCs using the microfluidic device is achieved by a series of steps.First, the enriched cell sample is exposed the replication-deficientadenovirus described within this document. This allows for the selectivelabeling of CTCs in the sample with a cell-surface, fluorescent fusionprotein also described herein. Once CTCs are labeled, as monitored byfluorescence intensity, the enriched sample is incubated with abiotinylated antibody that recognizes the fluorescent fusion proteinexpressed by CTCs and bind to these proteins at the surface of CTCs.After decorating CTCs with biotinylated antibody, enriched sample isflowed through the microfluidic device. Since the capture channels,including the microcolumns within them, have been functionalized with abiotin-binding protein (e.g., streptavidin), flowing cells bound withbiotinylated antibody, mostly CTCs, get trapped at the surfaces of thechannels. This trapping event is mediated by the strong covalent bondformed between the biotin-binding protein immobilized to the channel'ssurface and the biotin moiety in the antibody attached to the flowingCTC. Finally, loosely trapped cells are removed from the capturechannels by washing them away. Hence, only cells trapped viaimmunocapture remain immobilized in the channels of the microfluidicdevice.

After immunocapturing CTCs are released by exposing them to eithertris(2-carboxyethyl)phosphine (TCEP), a water-soluble, thiol-freereducing agent or to d-biotin depending on the biotin labeling systemused and by driving them out of the microfluidic device and into acollection microcentrifuge tube by laminar flow. Bath TCEP and D-biotinpreserve cell viability and their phenotypic characteristics. By usingeasy-release, biotinylated antibodies to immunocapture CTCs, this novelmethodology makes possible the favorable release of captured CTCs fromthe microfluidic device for effective cell culture propagation andmolecular characterization of the cells.

A microfluidic device is described herein which facilitates theefficient isolation of circulating tumor cells (CTCs) from an enrichedsample. This device is particularly well suited for use with the methodof the invention. However, it may also be adapted for use in othermethods as well.

A microfluidic device for isolating circulating tumor cells from asample is composed of an optically clear substrate having bound theretoat least four capture channels. In one embodiment, the substrate isglass and may be e.g., a standard microscope slide. Microscope slidesare usually made of glass, such as soda lime glass or borosilicateglass, but specialty plastics are also used. Fused quartz slides areoften used when ultraviolet transparency is important, e.g. influorescence microscopy. A standard glass slide typically measures about75 mm by 25 mm (3″ by 1″) and is about 1 mm thick. However, a range ofother sizes are available for various special purposes, such as 75×50mm, 46×27 mm, and 48×28 mm for thin sections. Slides are usually made ofcommon glass and their edges are often finely ground or polished.

A microfluidic device contains at least four, 6, 8, or more capturechannels comprising an arrangement of different-shaped microcolumns.These microcolumns include one or more cylindric, elliptical, triangularand chevron like microcolumn capture units. Each of the capture chamberscomprise at least floor and sidewall surfaces which have a capture agentfor circulating tumor cells in the sample. The device further containsan inlet reservoir and an outlet reservoir and distribution channels (5)which transport the flow of a sample from the inlet reservoir over thelength of the capture channels to the outlet reservoir.

The number of channels may be varied, e.g., from 4 up to about 8channels could be incorporated into the device based on size limits forthe device and the channels themselves. The arrangement of the capturechannels may vary from that of FIG. 2. For example, when the units arearranged in horizontal rows (perpendicular to the channel's walls), theycan also shift after every other or every third row. They can also beshifted vertically by varying the distance between neighboring rowsevery other or every third rows. Besides being arranged in horizontalrows, these units could also be organized in vertical rows (parallel tochannel's walls) and be shifted either horizontally or vertically asdescribed for the horizontal rows. Units could also be arranged randomlythroughout the channel. Alternatively, the channels may be fragmentedinto four shorter structures, each containing the differentmicrostructures, or connected in series to make up the full length of asingle channel presented in FIG. 2.

The desired measurements and patterns for the distribution of any of thecapture units, microcolumns and channels described herein are achievedby getting a photomask printed with this specific measurements anddesign. This design (along with measurements) is created using asoftware for Computer-Aid-Design (CAD) such as AutoCAD®. The softwarefile containing the design patterns of the invention are used to makeprint the photomask a described below. The photomask is used to make theSU-8 mold during the PDMS double casting prototyping by thermal aging ofPDMS.

With reference to FIG. 2, the device may have four capture channels (9).The capture channels are connected to an inlet (1) and an outlet (3) bya system of distribution channels (5), Each capture channel 9 containsan array of different-shaped microcolumns 13 that span the height of thechannel. These microcolumns 13 are organized within the channel incapture units that consist of either a set of hexagonally arrangedcylindrical microcolumns (FIG. 5A-D), a set of combined triangular andchevron-like microcolumns (FIGS. 7A-8C) or individual ellipticalmicrocolumns (FIGS. 6A-6D). Additionally, the capture channels and themicrostructures within them are chemically functionalized with thebiotin-binding protein avidin or its analogs for mediating the isolationof CTCs decorated with biotinylated antibodies against specific cellsurface epitopes prior to their introduction to the device. Thedifferent capture units within the channels are designed to disrupt thelinear path of flowing cells and to increase capture surface area; thus,maximizing the interaction between biotinylated antibody-coated cellsand the capturing agent. This approach to CTC isolation takes advantageof the rapid binding kinetics characteristic of the biotin-avidininteraction; therefore, lowering the duration of cell tosurface/microcolumn contact required to ensure cell capture.Furthermore, this approach facilitates the use of immunologic cocktails,which could help capture CFCs based on the presence of different cellsurface epitopes. This reduces or eliminates the dependency of CTCcapture on the expression of a single epitope.

The microfluidic device is designed with a system of distributionchannels that connect an inlet reservoir 1 to the capture channels forsample introduction and the capture channels to an outlet reservoir 3for sample removal, Suitably, the device is made of polydimethylsiloxane(PDMS) cast bonded to a glass slide 11 and FIGS. 3F-3I. Based onphysical properties, including transparency and porosity (for gasexchange) and biocompatibility, PDMS is particularly well suited as theelastomer. However, PMMA (poly(methyl methacrylate) may be selected andapplied to the substrate using methods such as hot-embossing rather thanphotolithography like PDMS. Engraved on the PDMS cast is the pattern ofgrooves and microcolumns that will form the characteristic channelsystem of the device upon bonding to the glass slide (FIGS. 3D-FIG. 3G).The PDMS double casting prototyping by PDMS thermal aging is the processby which the pattern of grooves and microcolumns is engraved on PDMS.

In one aspect of the device, the PDMS cast is produced by double castinga prototype combined with thermal aging of PDMS (FIG. 3E). This processfacilitates the appropriate replica of the microcolumns embedded in thecapture channel grooves. In this process, an SU-8 master containing thepattern for the channel grooves and microstructures is produced byphotolithography (FIG. 3E). Briefly, the designed pattern of channelsand microstructures for our device is transfer onto a photomask. Thismask is a glass plate containing the designed pattern printed on itssurface using an opaque material (chrome). The photomask is then broughtinto contact with a thin film (˜55 μm) of SU-8 (negative photosensitivesubstrate) previously deposited onto a silicon wafer. UV light is shownthrough the mask and onto the SU-8 film. Since SU-8 is a negativephotoresist, areas exposed to UV-light get crosslinked and solidify,while unexposed areas remain soluble. Unexposed SU-8 areas are thenremoved by washing the thin film with a developing solution, Thus, uponcompletion of the photolithography process, the desired pattern ofgrooves and microstructures is engraved on the SU-8 film, which is thenused as a mold to transfer this pattern onto the first PDMS replica(FIG. 3D).

Polydimethylsiloxane (PDMS) casting is achieved by mixing the PDMSpre-polymer with curing agent at a weight ratio of about 1:5 to about1:15, or about 1:8 to about 1:10, or about 1:9 (curing agent: base) andcuring these mixture at about 60 C for about 24 hours to about 80 C forabout 2 to 4 hours. Typically, higher temperatures within this range areassociated with the shorter curing times and the lower temperatures withlonger curing times, e.g., about 70° C. for about 3 to 4 hours, about80° C. for about 2 to 3 hrs or about 65° C. overnight (˜16 hrs). Thepre-polymer and curing agent are available commercially, e.g., asSYLGARD 184 silicone elastomer base & dimethyl, methylhydrogen siloxanecuring agent from Dow Corning. This pre-polymer to curing agent weightratio allows for fabrication of microstructures with the desiredelasticity to avoid damaging the patterned microcolumns during demoldingof PDMS casts. The aforementioned weight ratio and curing conditions areused during both PDMS castings steps. Then negative PDMS replicaresulting from the first casting process is then thermally aged (FIG.3G) at about 80° C. for at least about 72 hours PDMS slabs canalternatively be thermally aged at about 100° C. for about 48 hrs. Othervariations in temperature and time within these two ranges will beapparent to one of skill in the art. Finally, the thermally agednegative PDMS replica serves as mold to cast a second PDMS replica withpositive orientation (FIG. 3H).

In another aspect of the device, microfluidic units are fabricated bybonding a positive PDMS cast or replica to a glass slide after surfaceactivation of both components by plasma edging (FIG. 3I). Bondedmicrofluidic units have width and length dimensions similar to a typicalmicroscope slide, 25 and 75 mm respectively. The height of the devicevaries depending on the volume of PDMS used during the second castingstep.

In one aspect of the device, the inlet and outlet reservoirs used have adiameter of 2 mm. The inlet reservoir is connected to the capturechannels entry side by distribution channels 55 μm in height. Asillustrated in FIG. 2, distribution channels may be composed of a short,vertical channel. In one embodiment, such a channel is about 1 mm inlength and about 1.5 mm in width. This channel connects to a longperpendicular channel 8.5 mm in length and 1 mm in width that splits thesample into two sample delivery sections. Each delivery sectiondistributes sample to two of the four capture channels through avertical channel 4 mm in length and 1.5 mm in width. This verticalchannel opens about 45° on each side to gradually increase thecross-sectional flow area and introduce the sample into both capturechannels included in the specific delivery section. A similar system ofdistribution channels facilitates the transport of sample from the exitside of the capture channels to the outlet reservoir for sample removal.

In yet another aspect of the device, capture channels are about 44 mm inlength, about 3 mm in width and about 50 microns (μm) to about 70microns height (depth). In one embodiment, the channels are about 55 μm.Each capture channel comprises an array of different-shaped microcolumnsthat include cylindrical, elliptical and a combination of triangular andchevron-like columns arranged in different orientations.

In one aspect of the device, the cylindrical microcolumns included inone of the channels have a diameter of about 100 μm to about 200 μm anda height of about 50 μm to about 70 μm. In one embodiment, the channelshave a diameter of about 100 μm and a height of about 55 μm. Thecylindrical microcolumns are arranged in hexagonal-shaped units whichunits about 340 μm to about 700 μm in width and length. In oneembodiment, the hexagonal-shaped units are about 370 μm in width andlength. Each hexagonal unit is comprised of seven individual cylindricalmicrocolumns distributed so that six microcolumns sit at each vertex ofthe hexagon and one microcolumn sits at the center of the hexagon. Theseunits may be arranged in rows which are horizontally or verticallylocated relative to the substrate. Typically, these rows ofhexagonal-shaped units are shifted horizontally or vertically after eachtwo to three rows. However, other shifts may be selected, or thehexagonal-shaped units may be randomly located. Individual cylindricalmicrocolumns are separated about 20 μm to about 50 μm, or about 35 μmfrom neighboring microcolumns within a hexagonal unit. Additionally,individual hexagonal units are separated by about 35 μm to about 75 μm,or about 50 μm within each row of units and each hexagonal unit isshifted about 35 μm to about 75 μm, or about 60 μm horizontally afterevery two to three rows. When in vertical rows, similar shifts may beapplied.

In another aspect of the device, two of the capture channels includecapture units that combine triangular and chevron-like microcolumns inheight. These capture units may range in size from about 210 μm to about700 μm. Each of these unique capture units is comprised of two paralleltriangular columns with a base and height (depth) of about 100 to about325 μm each and a separation from about 25 μm to about 50 μm betweenthem. A chevron-like column placed at the base of the paralleltriangular microcolumns complements these capture units. Thesechevron-like columns are about 225 μm in to about 700 μm, or about 250μm in length, about 20 μm to about 50 μm in width and have an aperatureangle of about 145° to about 165°, or about 150° aperture angle. Inaddition, the orientation of the chevron-like columns serves todifferentiate the capture units on each of the two channels containingthis array. Capture units in which the chevron-like microcolumn pointstoward the base of the parallel triangular columns, the minimumseparation between chevron-like and triangular columns is about 20 μm toabout 50 μm, or about 35 μm, and this type of capture unit is about 200μm to about 700 μm, or about 210 μm in length and about 200 μm to about700 μm, or about 250 μm in width. The combined units where thechevron-like column has the opposite orientation, that is it points awayfrom the base of triangular columns, the minimum separation between thechevron-like and triangular columns is about 20 μm to about 50 μm andthe complete capture unit is 200 μm to about 700 μm in length and width,Each of these types of capture units may be separated from neighboringindividual units within and between rows, and are shifted 200 μmhorizontally or vertically every row, every other Tow, or the units maybe located randomly.

As with the hexagonal shaped capture units composed of cylindricalcolumns, the distances from unit to unit within a row or from row to roware not critical. These capture units can also be arranged in horizontalrows (perpendicular to channel's walls) and shift horizontally but afterevery other or every third row. They can also be shifted vertically byvarying the distance between neighboring rows every other or every thirdrows. Besides being arranged in horizontal rows, these units could alsobe organized in vertical rows (parallel to channel's walls) and beshifted either horizontally or vertically as described for thehorizontal rows. Finally, units could also be arranged randomlythroughout the channel. In one aspect of the device, ellipticalmicrocolumns embedded in the last of the four channels have dimensionsof about 100 μm to about 250 μm, or about 150 μm and about 50 μm toabout 150 μm for major and minor axes and height. In one embodiment, themajor and minor axes are about 75 μm or about 55 μm respectively. Thedistance between single units and between rows of units may be varied.In one example, each elliptical microcolumns is separated by about 50 μmto about 70 μm from its neighboring columns within a row and about 50 μmto about 70 μm, or about 35 μm from neighboring columns between rows.Elliptical microcolumns shift from about 100 μm to about 250 μmhorizontally or vertically after every row, or every other row, whenarranged in rows and not randomly spaced in the channel.

Once produced, the device can be used to detect circulating tumor cellsas described herein. Alternatively, the device may be adapted for use inother methods.

In one aspect, sample suspected of containing circulating tumor cells isintroduced into the inlet reservoir of the device. In one embodiment,sample suspected of containing circulating tumor cells is introducedinto the device via a flexible tube (e.g., Tygon® tubing, St. Gobain),which connects the source containing the sample (e.g., a metal hub bluntpoint needle fixed to a syringe) to the inlet reservoir of the device.Similar tubing is also attached to the outlet reservoir for outflow intoa waste or collection container, typically a microcentrifuge tube. Thesample is injected into the device with a continuous flow at ratesranging from about 10 μl/min to about 30 μmin. Once the sample has beeninjected, the channels are washed from two to three times with asuitable solution (e.g., a phosphate buffered saline (PBS)) with acontinuous flow at rates ranging from about 50 μl/min to 200 μl/min. Inone embodiment, a syringe pump or an equivalent means capable ofproducing flow rates in the μl/min range is used to control the flow forsample injection and washing steps. A suitable amount of the sample ispermitted to flow through the distribution channel into the verticalchannels and all surfaces of each capture channel come in contact withthe flowing sample. The surfaces, which include the floor, ceiling, andsidewalls of the capture chambers of the microcolumns within eachchannel, are coated with a suitable capture agent. For use in connectionwith circulating tumor cells, the capture agent may be a biotin-bindingagent selected from a group of biotin-binding proteins including avidin,streptavidin and deglycosylated avidin (such as NeutrAvidin™).

The capture agent may be bound directly or indirectly to the surface.For example, in one embodiment, the capture agent is immobilized to allcapture surfaces of each channel via a cross-linker. Suitablecross-linkers may include N-[γ-maleimidobutyryloxy]succinimide ester(GMBS), which is used to covalently attach the capture agent channelsurfaces. Covalent attachment of the capture agent is facilitated by theN-hydroxysuccinimide ester moiety of GMBS. In another embodiment, thecapture agent is covalently attached to all capture surfaces of eachchannel through the maleimide moiety of the cross-linker GMBS. Priorsilanization of capture surfaces is required for the covalent attachmentof the cross-linker. The silanization agent(3-mercaptopropyl)trimethoxysilane may be used for surface activation onthis device. Still other capture agents may be selected for use in thepresent invention.

The words “comprise”, “comprises”, “comprising”, “contain”, “contains”and “containing” are to be interpreted inclusively rather thanexclusively. The works “consist”, “consisting”, and its variants, are tobe interpreted exclusively, rather than inclusively.

As used herein in reference to numeric values provided herein, the term“about” may indicate a variability of as much as 10% unless otherwisespecified.

The following examples are illustrative of embodiments of the inventionand do not limit the scope of the invention.

Example 1

Example 1 describes preparation and process of patient-derived bloodsamples for CTC Analysis, i.e., CTC-enrichment which is optionally usedin conjunction with the non-lytic adenoviral system for detection ofcirculating CTC.

A. Initial Sample Processing

After discarding initial two 6 mL tube collections to avoidcontamination by epithelial stem cells, peripheral blood samples will beobtained in two 6-mL sodium heparin green top tubes and immediatelyplaced on ice. Blood will be drawn until vacuum ends, with ideal totalblood volume of at least 10 mL. Specimens will be processed within twohours of collection in compliance with CTC enrichment protocolmanufacturer recommendations.

B. CTC Enrichment

Following combination of 10 mL sterile phosphate buffered saline inconical with blood collection, mixture will be chilled on ice for 15minutes. Meticulous care will be exercised to introduce blood solutionto OncoQuick tube, prior to centrifugation for 20 minutes at 1500 RCF.

Harvesting of the CTC-enriched layer consists of upper plasma layeraspiration to 5 mL above the demarcated interphase layer. The remainingsolution overlying the porous barrier will be sterilely extracted andplaced in a separate 50 mL conical containing 30 mL sterile wash buffer(0.5% Bovine Serum Albumin in 1×PBS). 10 additional mL wash buffer willbe introduced to Onco-quick tube to collect residual CTCs adhering toconical or porous barrier surfaces, before adding to the separate 50 mLconical, bringing total CTC enriched dilute solution to 50 mL totalvolume. Centrifugation of this conical will be conducted for 10 minutesat 250 RCF. Aspiration to 5 mL gradation will be carefully performed,prior to employment of P-1000 mcL pipettor until 500 mcl CTC enrichedcell pellet suspension remains. Following incubation in 37° C. waterbath, 1000 mcl cell culture media (DMEM, 10% fetal bovine serum, 1%Penicillin/Streptomycin) will be introduced to allow cell pelletre-suspension, prior to aliquot of 750 mcl into each of twopoly-D-lysine chamber wells.

B. Assay:

CTC Assay Protocol: Materials and Methods

Materials:

-   -   Gradient centrifuge tube (e.g. Oncoquick tube)    -   50 mL propylene centrifugation tube    -   Centrifuge capable of generation 1600 RCT at 4° C. with swinging        bucket rotor and tube carriers/adapters for 30×115 mm tube size    -   Disposable serological pipettes    -   Washing buffer: 1× phosphate buffered saline (PBS)+0.5% w/v        bovine serum albumin (BSA)    -   Gloves appropriate for protection vs bloodborne infections    -   Blood sample    -   Poly d lysine coated chamber wells and 6 cm petri dish

1. Introducing Patient Blood Sample into Tube (See, e.g., FIG. 1A)

-   -   To minimize further possible loss of tumor cells due to        non-specific adsorption to dry plastic surfaces, prewet all dry        surfaces with washing buffer by simply pipetting up and down        once or rinsing once to prewet all fresh serological pipettes or        50 mL tubes respectively    -   Do not introduce blood right onto porous barrier since this can        result in mix up the separation medium with blood thus        decreasing separation quality    -   Slightly incline the oncoquick tube and introduce blood slowly        down side of tube    -   Preparation        -   Precool centrifuge to 4° C.    -   Obtain ice bucket filled with ice        -   Precool oncoquick tubes and blood speciments for 10-15 min            on ice (important to perform this procedure within 2 hours            of blood draw)        -   Ensure separation medium (blue) is completed in the lower            compartment and if not-->spin oncoquick tube to bring            separation medium back into lower compartment        -   If oncoquick solution is higher than porous            line-->centrifuge on RCF slow 1500 RCT for 4 min        -   Obtain mask, gown, gloves        -   Blood sample contained in 2 separate 6 mL tubes containing            sodium heparin        -   Spray down blood sample tubes with EtOH    -   Enrichment: washing steps can be performed at RT if convenient    -   Prewash conical with wash buffer (add 25 mL 0.5% BSA in PBS and        remove)    -   Aliquot:    -   Sporogon into 50 mL conical    -   0.5% BSA in PBS (wash buffer used to wash any pipette that will        make contact with cells to prevent adherence to conical) into 50        mL conical    -   Sterile PBS into 50 mL conical    -   Obtain 10 mL pipette and wash (×1) inside of 10 mL pipette with        wash buffer    -   Pipet 10 mL sterile PBS and place in 50 mL conical    -   Obtain 10 mL pipet and wash with wash buffer    -   Invert blood sample×5 and repeat with collection of all blood        from both tubes into one 10 mL pipet    -   Make note of total blood volume (around 10 mL)    -   Add blood sample (approximately 5 mL usually from each blood        tube sample) to 10 mL sterile PBS-->pipet×5 for adequate mix        ensuring not to advance higher than blood line    -   Wash pipet with sporogon (×2) and discard    -   Incubate oncoquick tubes and blood specimens on ice for 15        minutes    -   Wash 25 mL pipette with wash buffer (×1)-->pipet blood-->obtain        all blood in pipette and carefully/slowly introduce in side of        oncoquick tube    -   Fill the cooled whole blood (15-30 mL) gently into upper        compartment without disturbing medium underneath porous barrier        via introducing to side of oncoquick tube    -   Note: use 25 mL pipet to obtain first collection then switch to        2 mL pipet to obtain remaining blood in conical to ensure least

amount of remnant cells

-   -   With anti-viral solution (e.g., Spor-Gon) disinfect the used        pipette and the remaining conicals

2. Centrifugation and Cell Separation (See, e.g. FIG. 1B)

-   -   Spin blood filled OncoQuick™ tube at 1500×g (aka RCF) and 4 C        for 20 min in a swing bucket rotor with slow acceleration and no        brake    -   After centrifugation, tumor cells will be in interphase between        upper plasma (yellow/brown) and lower separation medium (blue).        Usually this cell fraction is not visible.    -   If interphase is at 20 or lower-->aspirate to 25, but if        interphase layer exceeds 20 mark then aspirate to 5 mL above        interphase layer    -   Place 30 mL wash buffer in fresh conical/centrifugation tube

3. Removal of CTC-Enriched Cell Layer (See, e.g., FIG. 1C)

-   -   The entire remaining liquid volume above the porous barrier can        be collected with sterile serological 10 mL pipette and        transferred to mix with 30 mL wash buffer    -   Remove top of the layer down to prevent most cells from sticking        to porous barrier    -   Carefully rinse inner tube wall and the surface of the porous        barrier of the emptied oncoquick tube with approximately 10 mL        washing buffer to collect cells eventually adhering to these        surfaces    -   Add them to centrifuge tube already containing transferred        liquid volume and bring volume to a total 50 mL using wash        buffer (had 10 mL sample and add 30 mL wash buffer, make total        50 mL by adding 10 mL wash buffer to oncoquick to retrieve        remaining cells)    -   Mix suspension by gently inverting tube 5 times    -   Pellet the cells at 250×g (aka RCF) for 10 min    -   Obtain 2 mL media (10% FBS, 1% Pen Strep) and place in water        bath warmer    -   Gently aspirate about 49.5 mL supernatant without disturbing        cell pellet and leave the pellet in the remaining 500 microL of        washing buffer    -   Perform this by aspirating to 5 mL mark, then obtain P1000 pipet        and aspirate 1 mL×4 then an additional 500 microL (each time        with new pipet tips)    -   Add 1 mL culture media (10% FBS. 1% Pen Strep)

3. Moving CTCs to Chamber Slide for Immunofluorescent Microscopy (See,e.g., FIG. 1D)

-   -   Obtain new tip and set to 750 microL-->add 750 microL (obtained        from middle of solution) to each well in 2 poly D lysine coated        chamber slide wells    -   Divide remaining fraction between both wells (do one drop at a        time)

4. Adding Vector (See, e.g., FIG. 1E)

-   -   Add 495 microL of media to 5 microL virus aliquot in aliquot        labeled “V”    -   Pipet up and down 5 times to mix    -   “1:100”        -   Obtain new pipet tip set to 50 microL and obtain 50 microL            solution from “V”, adding to new aliquot labeled “1:100”        -   Add 450 microL media (10% FBS, 1% Pen Strep) to separate            aliquot labeled 1:100        -   Mix thoroughly×5        -   Put 50 microL of diluted OBP 401 in 1:100 well    -   Using new gloves, remove chamber well from under hood and place        in petri dish, covering with clean gloved hand    -   Incubate chamber well in 37° C. incubator

F. Method for Verification and Quantification of CTCs (See, e.g., FIG.1F)

CTCs may be imaged, quantitated, and characterized. Fluorescentmicroscopy imaging can be used as follows. At 24 hours posttransfection/infection, 100× tiled fluorescent images of each chamberwell will be obtained, prior to fixation to avoid potential loss of nonpoly-d-Lysine adherent, circulating tumor cells. Images will be savedand subsequent analysis per semi-automated computer software will beperformed to allow sorting, capturing and filtering of CTC. Enumerationwill then be employed per established parameters (Fluorescent intensity,cell area, cell diameter) and algorithm. Following imaging, chamberwells will be fixed with 10% neutral buffered formalin solution.Counterstaining can then be performed to qualitatively assess isolatedcirculating tumor cells.

Example 2 A Telomerase-Based Single-Adenovirus Assay Detects CirculatingTumor Cells

Human glioblastoma-derived U251 cells were obtained from the AmericanType Culture Collection, ECACC catalogue number: 09063001 and suspendedin Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad,Calif.) supplemented with 10% fetal bovine serum (FBS, Invitrogen,Carlsbad, Calif.) and 1.0% penicillin-streptomycin at 37° C. in anatmosphere of 5% CO₂. Cells were seeded on 8 well Poly-d-lysine chamberslides at a pre-determined optimal density of 5×10³ cells/well(enumeration provided via Nexcelom Biosciences' Cellometer in duplicate)just prior to viral infection. A non-lytic adenoviral particle accordingto the invention was constructed using conventional techniques, e.g., asdescribed on http://www.med.upenn.edu/gtp/vectorcore.production.shtml.The source of adenoviral capsid and genomic sequences was humanadenovirus 5 (Ad5).

The Human TERT promoter was synthesized based on the sequences publishedpreviously (Kawashima, T, et al. Telomerase-SpecificReplication-Selective Virotherapy for Human Cancer. Clin Cancer Res.,Jan. 1, 2004 10; 285). The transgene expression cassette containing theHuman TERT promoter was first inserted in pShuttle plasmid and flankedin between 2 meganuclease enzyme sites, I-CeuI and PI-SceI. Theexpression cassette was then excised by I-CeuI and PI-SceI digestion andsub-cloned into adenovirus backbone plasmid (pH5′0.040.CMV.A1AT fromPenn Vector Core) by replacing the fragment flanked by the same enzymes.The recombinant adenoviral vector is generated by treating the plasmidwith PacI to release both Inverted Terminal Repeat sequences (ITRs) ofthe adenoviral vector genome, followed by transfection (e.g. 5 μgs per25 cms flask) and rescue in HEK293 cells. The vectors were expanded andpurified by density gradient centrifugation from the lysate harvestedfrom infected 293 cells via by 3 cycles of freeze-thawing. The physicalparticle titer of adenoviral vectors was determined using absorbancereadings at 260 nm via a spectrophotometer. The vectors were furthercharacterized by genome DNA structure analysis, infectivity assay andreplication competent adenovirus (RCA) assay. See, e.g.,http://www.med.upenn.edu/gtp/vectorcore/quality_control.shtml.

In the resulting construct, termed Ad.hTert.eGFP contains deletions inthe wild-type E1 region (E1a and E1b) and the E3 region of the Ad5,rendering it non-lytic. The construct further contains an exogenousexpression cassette containing, from 5′ to 3′ the hTERT promoter (376bp+5′ UTR). eGFP gene, a polyA-Woodchuck hepatitis viruspost-transcriptional regulation element (WPRE), and a rabbitbetaglobulin poly A. Use of the hTERT promoter makes the expression ofthe enhanced green fluorescent protein dependent upon the presence oftelomerase, which is indicative of the presence of circulating tumorcells. This adenovirus does not contain any insert in the E3-deletedregion, and with the exception of the E1-deletion and E3-deletionretains other adenoviral genomic regions necessary for to package thegenomic sequences into the adenoviral capsid and form thereplication-defective adenoviral particle. The techniques used togenerate the vector are well known to those of skill in the art.

Following titration of the cells into the wells, Ad.hTert.eGFP viralparticles of non-lytic telomerase-specific adenoviral vector were addedto the cells at various concentrations (5.9×10⁷′5.9×10⁸, 2.95×10⁸,2.95×10⁹, 1.1×10¹⁰ viral particles (also termed genome copies)), whichare thereafter incubated at 37° C. under 5% CO₂. Expression levels ofeGFP protein in the cells are tested at 24, 28, 72, 96, and 120 hours.In order to maintain cell viability during this time, media is replacedas needed.

Fluorescent cells were detected via a PlanNeofluor lens objectivemounted on a Nikon TE-2000 microscope equipped with epifluorescenceoptics. The epifluorescent images were captured with a Hammamatsucharge-coupled-device camera that was controlled with IP LabSpectrumsoftware, thus detecting cells expressing GFP.

These results showed that a dilution of 1:100 was optimal, showingapproximately 3 billion viral particles and good imaging at 48 hours.However, this system using the non-lytic telomerase-specific adenoviralvector still showed acceptable results over all time points and at allconcentrations.

Example 3 A Telomerase-Based Two Adenovirus Assay Detects Circulating

Tumor Cells

Human glioma cells U251 and suspended in a suitable as described inExample 2 above. Cells were seeded on 8 well Poly-d-lysine chamberslides at a pre-determined optimal density of 5×10³ cells/well(enumeration provided via Nexcelom Biosciences' Cellometer in duplicate)just prior to viral infection.

A first non-lytic adenoviral particle according to the invention wasconstructed and is termed hTert.Cre. The source of adenoviral capsid andgenomic sequences was human adenovirus 5 (Ad5). In the resultingconstruct, the wild-type E1 region (E1a and E1b) and the E3 region ofthe Ad5 are deleted. An exogenous expression cassette containing, from5′ to 3′ the hTERT promoter, the Cre recombinases coding sequence, apolyA-Woodchuck hepatitis virus post-transcriptional regulation element(WPRE). This adenovirus does not contain any insert in the E3-deletedregion, and with the exception of the E1-deletion and E3-deletionretains other adenoviral genomic regions necessary for to package thegenomic sequences into the adenoviral capsid and form thereplication-defective adenoviral particle. A second adenoviral particle,termed Ad.CB.Flex.eGFP was also constructed. This construct contains anE1- and E3-deletion, and an exogenous expression cassette in the site ofthe E1 deletion. The expression cassette contains a CAGS promoter, aloxP site, an inverted eGFP gene—polyAWPRE, and a second loxP sequence.The CAGS promoter is a ubiquitous strong promoterienhancers composed ofthe chicken β-actin promoter with human cytomegalovirus immediate early(CMVIE) enhancer. The techniques used to generate the vector are wellknown to those of skill in the art. In this two-vector system,telomerase-specific expression of the Cre protein caused the invertedreporter gene to flip and expression the fluorescent protein. Thissystem is self-amplifying because the CAGS promoter is constitutive.

Following titration of the cells into the wells, the two non-lytictelomerase-specific adenoviral vectors were added to the cells at1.14×10⁹ combined viral particles or 1.14×10⁸ combined viral particles(genome copies or GC). Because this system is self-amplifying, theration of Ad.hTert.Cre to Ad.CB7.Flex.eGFP is 2:1. Following infection,the cells are incubated at 37° C. under 5% CO₂. Expression levels ofeGFP protein in the cells were tested at 24, 28, 72, 96, and 120 hours.In order to maintain cell viability during this time, media was replacedas needed.

These results show that the combined virus system, while expression timewas somewhat slower than the single virus system of Example 2, higherexpression levels were obtained.

However, this system using this dual non-lytic telomerase-specificadenoviral vector system, acceptable results were observed.

Example 4 Microfluidics Device for Cell Capture

A device was produced from a PDMS cast prepared by double castingcombined with thermal aging of PDMS. PDMS pre-polymer and curing agentused to make this device are Sylgard® 184 Silicone Elastomer Kit(Ellsworth Adhesives). This process facilitates the appropriate replicaof the microcolumns embedded in the capture channel grooves. In thisprocess, an SU-8 master of positive orientation was produced byphotolithography first. This SU-8 master then served as mold to cast thefirst PDMS replica with negative orientation. PDMS casting was achievedby mixing the PDMS pre-polymer with curing agent at a weight ratio of1:9 and curing the mixture at 70° C. for 3 to 4 hours. The same weightratio and curing conditions were used during this and the subsequentPDMS castings. The negative PDMS replica resulting from the firstcasting process was then thermally aged at 80° C. for at least 72 hours.Finally, the thermally aged negative PDMS replica served as a mold tocast a second PDMS replica with positive orientation. Since PDMSpre-polymer is too viscous, volumes are actually measured by weighingthe material under the assumption that 1 g=1 ml. Thus, 60 g of Sylgard®184 Silicone Elastomer pre-polymer and 6.66 g of Sylgard@184 SiliconeElastomer pre-polymer were measured, mixed, cast and cured. The heightof the device once the PDMS slab was bonded to a glass slide was ˜4 mm.

The microfluidic units are fabricated by bonding the positive PDMSreplica to a glass slide after surface activation of both components byplasma edging. The bonded microfluidic units had width and lengthdimensions similar to a typical microscope slide, 25 and 75 mmrespectively. The glass photomask used to produce the SU-8 master byphotolithography during the process described for PDMS double castingprototyping by thermal aging of PDMS contains, printed in chrome, thedesigned pattern of channels and microstructures with the specifieddimensions. That is, each pattern or structure on the design is printedon the surface of the glass photomask with the specified dimensions.

The inlet and outlet reservoirs have a diameter of 2 mm. The inletreservoir is connected to the capture channels entry side bydistribution channels 55 μm in height. Distribution channels were short,vertical channels 1 mm in length and 1.5 mm in width. This channelconnects to a long perpendicular channel 8.5 mm in length and 1 mm inwidth that splits the sample into two sample delivery sections. Eachdelivery section distributes sample to two of the four capture channelsthrough a vertical channel 4 mm in length and 1.5 mm in width. Thisvertical channel opens about 450 on each side to gradually increase thecross-sectional flow area and introduce the sample into both capturechannels included in the specific delivery section. A similar system ofdistribution channels facilitates the transport of sample from the exitside of the capture channels to the outlet reservoir for sample removal.

The capture channels are 44 mm in length, 3 mm in width and 55 μm inheight. Each capture channel comprises an array of different-shapedmicrocolumns that include cylindrical, elliptical and a combination oftriangular and chevron-like columns arranged in different orientations.

The cylindrical microcolumns included in one of the channels have adiameter of 100 μm, a height of 55 μm and are arranged inhexagonal-shaped units of 370 μm in width and length. Each hexagonalunit is comprised of seven individual cylindrical microcolumnsdistributed so that six microcolumns sit at each vertex of the hexagonand one microcolumn sits at the center of the hexagon. Individualcylindrical microcolumns are separated 35 μm from neighboringmicrocolumns within a hexagonal unit. Additionally, individual hexagonalunits are separated 50 μm within each row of units and each hexagonalunit is shifted 60 μm horizontally after every row.

Two of the capture channels include capture units that combinetriangular and chevron-like microcolumns 55 μm in height. Each of theseunique capture units is comprised of two parallel triangular columnswith a base and height of 100 μm each and a separation of 50 μm betweenthem. A chevron-like column placed at the base of the paralleltriangular microcolumns complements these capture units. Thesechevron-like columns are 250 μm in length, 50 μm in width and have a150° aperture angle. In addition, the orientation of the chevron-likecolumns serves to differentiate the capture units on each of the twochannels containing this array. Capture units in which the chevron-likemicrocolumn points toward the base of the parallel triangular columns,the minimum separation between chevron-like and triangular columns is 35μm and this type of capture unit is 210 μm in length and 250 μm inwidth. Differently, combined units where the chevron-like column has theopposite orientation, that is it points away from the base of triangularcolumns, the minimum separation between the chevron-like and triangularcolumns is 50 μm and the complete capture unit is 250 μm in length andwidth. Furthermore, both of these types of capture units are separated50 μm from neighboring individual units within and between rows, and areshifted 200 μm horizontally every row.

The elliptical microcolumns embedded in the last of the four channelshave dimensions of 150 μm, 75 μm and 55 μm for mayor and minor axes andheight respectively. Each elliptical microcolumns is separated 50 μmfrom its neighboring columns within a row and 35 μm from neighboringcolumns between rows. Elliptical microcolumns shift 100 μm horizontallyafter every row.

For cell capture, surfaces of the capture channels of the device thatcome in contact with the flowing sample are coated with capture agent.

Example 5 Use of Microfluidics Device for Capturing CTC Using Assay ofMethod

All surfaces of each capture channel of the device prepared according toExample 4 were coated with capture agent. The capture agent used in thisinstance consisted of deglycosylated avidin (NeutrAvidin™). Channelsurfaces were coated with 10 ng/ml of deglycosylated avidin(NeutrAvidin™) by first injecting 900 μl of a 4% (v/v)(3-mercaptopropyl)trimethoxysilane solution into the device channels andincubating it 45 minutes at room temperature (22° C.). After washing thechannels with 2 ml of 100% ethanol, 900 μl of a 0.28% (v/v) GMBSsolution was injected into the device and the device was incubated for35 min at room temperature. The channels were washed again with 2 ml of100% ethanol followed by 2 ml of ultrapure water and 2 ml of 1×Phosphate Buffered Saline (PBS) solution. Finally, 900 μl of a 10 ng/mlsolution of deglycosylated avidin (NeutrAvidin™) were injected into thechannels of this device. The device was then stored at 4° C. with itschannels filled with the deglycosylated avidin solution until needed.One hour before running a sample through the device, channels wereflushed with 2 ml of 1×PBS followed by 1 ml of a 1× Phosphate BufferedSaline (DPBS), 1% (w/v) Bovine Serum Albumin (BSA) and 0.09% (w/v)Sodium Azide. All solutions, including washing solutions, were injectedwith a continuous flow at a rate of 500 μl/min controlled by a NANOmitesyringe pump (Harvard Apparatus).

For a proof of principle demonstration of the combined detectionprocess, 5×10⁴ U251 cells were incubated with 5×10⁹ GC/ml ofAd.hTert.pCherry.WPRE.RBG vector for 48 hours at 37° C. with 5% CO₂.Most of the cells incubated with the vector were intensively fluorescingafter the 2-day incubation period (FIGS. 9A-9B). Cellular fluorescencewas an indicator that the infected cells were producing the redfluorescent fusion protein (pCherry) encoded by the vector's genome. Tomimic the composition of an enriched sample, the infected U251 cellswere mixed with Jurkat cells (not exposed to vector) at a ratio in whichinfected U251 cells represented 1% of the total cell content of thesample. This cell mixture was then supplemented with biotinylatedAnti-pCherry at a final concentration of 10 μg/ml, brought up to a finalvolume of 500 μl with a 1× Phosphate Buffered Saline (DPBS), 1% (w/v)Bovine Serum Albumin (BSA) and 0.09% (w/v) Sodium Azide solution andincubated on ice for 30 minutes. After the incubation, the volume of thesample was adjusted to 1 ml with plain 1×PBS solution and loaded into a1 ml syringe. 850 μl of the sample were injected into one of our devicespreviously functionalized with declycosylated Avidin (NeutrAvidin™) andflushed with 1× Phosphate Buffered Saline (DPBS), 1% (w/v) Bovine SerumAlbumin (BSA) and 0.09% (w/v) Sodium Azide solution as described withinthis document. The sample was injected with a continuous flow at rate of15 l/min controlled by a NANOmite syringe pump (Harvard Apparatus). Uponcompletion of sample injection, device channels were washed twice with 1ml of IX PBS to remove cells loosely bound to the channels/microcolumns.To be able to assess purity of the captured sample, the nucleus oftrapped cells was stained with the fluorescent dye Hoechst 33342. Thiswas achieved by injecting 400 μl of a 0.5 μM Hoechst 33342 solutionimmediately after the second IX PBS wash and incubating the device for30 minutes at room temperature. After the incubation with Hoechst, thechannels of the device were tiled (FIGS. 10A-10C) and captured cellswere analyzed using the Eclipse TE2000-U fluorescent microscope (NikonCorp.) equipped with an automated stage and image analysis programsdriven by Image-Pro Plus 7.0 (Media Cybernetics). Representative stillpictures of captured cells were also collected under the red (pCherry)and blue (Hoechst) channels as well as under brightfield (FIGS.11A-11C).

Preliminary data suggests that the rough capture efficiency is about 96%on average. This number is based on the average of three single countsof the flow-through samples collected after the injection of either5.5×10³, 4×10⁴ or 8.5×10⁴ U251-infected cells (pCherry positive) mixedwith 1×10⁶ Jurkat cells and 10 g/ml biotinylated Anti-pCherry into threeseparate devices at a continuous flowrate of 15 μl/min. The flow-throughsamples were analyzed and counted using an automated cells counter withfluorescence capabilities (Cellometer Vision, Nexcelom). This instrumentcounts a 20 μl fraction of the flow-through sample and providesconcentrations for the total number of cells found in the sample, bycounting under bright field mode and the number of red fluorescent cellsby counting cells under a red optic channel. These concentrations wereused to indirectly calculate the total number of cells captured based onthe number of U251 cells found in the flow-through sample (FIG. 12).Capture efficiency and purity of can be assessed using tiled imagescollected from the Eclipse TE2000-U fluorescent microscope (Nikon Corp.)and the image analysis programs Image-Pro Plus 7.0 (Media Cybernetics).

All publications cited in this specification are incorporated herein byreference, as is priority application U.S. 61/718,993, filed Oct. 26,2012. While the invention has been described with reference toparticular embodiments, it will be appreciated that modifications can bemade without departing from the spirit of the invention. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A microfluidic device for isolating circulating tumor cells from asample, wherein the device comprises: (a) an optically clear substratehaving bound thereto at least four polydimethylsiloxane-based capturechannels (9), wherein each of the capture channels (9) comprise anarrangement of different-shaped microcolumns (13) selected from thegroup of one or more of cylindric, elliptical, triangular andchevron-like microcolumn capture units, and wherein each of themicrocolumn capture chambers comprise at least floor and sidewallsurfaces which have a capture agent for circulating tumor cells in thesample; (b) an inlet (1) reservoir and an outlet reservoir (3); (c)distribution channels (5) which transport the flow of a sample from theinlet reservoir (1) over the length of the capture channels (9) to theoutlet reservoir (3).
 2. The microfluidic device according to claim 1,wherein the optically clear substrate is glass.
 3. The microfluidicdevice according to claim 1, wherein the microcolumn capture unitsfurther comprise a capture agent-bound ceiling.
 4. The microfluidicdevice according to claim 3, wherein the capture agent is abiotin-binding agent.
 5. The microfluidic device according to claim 4,wherein the biotin-binding agent is selected from avidin, streptavidinand a deglycosylated avidin.
 6. The microfluidic device according toclaim 1, wherein each of the microcolumn cylindrical, elliptical,triangular and chevron-like capture chambers are of essentiallyidentical depth or height.
 7. The microfluidic device according to claim6, wherein the capture chambers can range in depth from about 50 micronsto about 70 microns.
 8. The microfluidic device according to claim 7,wherein the capture chambers are about 55 microns in depth.
 9. Themicrofluidic device according to claim 1, wherein the cylindricalmicrocolumn capture units are arranged in hexagonal-shaped unitscomprising about seven cylindrical capture chambers.
 10. Themicrofluidic device according to claim 9, wherein the hexagonal-shapedunits are arranged in horizontally or vertically rows relative to thesubstrate.
 11. The microfluidic device according to claim 10, whereinsaid rows of hexagonal-shaped units are shifted horizontally orvertically after each two to three rows.
 12. The microfluidic deviceaccording to claim 9, wherein the cylindrical microcolumn capturechambers have a diameter of about 100 μm to about 200 μm, a height ofabout 50 μm to about 70 μm and the hexagonal-shaped units are about 340μm to about 700 μm in width and length.
 13. The microfluidic deviceaccording to claim 1, wherein two of the capture channels comprise aplurality of capture units which comprise a pair of parallel triangularcapture chambers and a chevron-like capture chamber, wherein in one ofthe capture channels the chevron-like capture chambers are oriented suchthat it points toward the base of the pair of parallel triangularchambers within a capture unit and in a second of the capture channelsthe chevron-like capture chamber is oriented such that it points awayfrom the base of the triangular chambers.
 14. The microfluidic deviceaccording to claim 13, which said each of said capture units comprisinga pair of parallel triangular capture chambers and a chevron-likecapture chamber has a width of about 210 μm to about 700 μm.
 15. Themicrofluidic device according to claim 13, wherein each of thetriangular capture chambers has a base and depth of about 225 μm toabout 700 μm in length and a separation of about 2.5 μm to about 50 μmbetween the triangular chambers.
 16. The microfluidic device accordingto claim 13, wherein the chevron-like capture chamber has a length ofabout 225 μm to about 700 μm, a width of about 50 μm to about 100 μm andan aperature angle of about 145° to about 165°.
 17. The microfluidicdevice according to claim 13, wherein there is about 20 μm to about 50μm between chevron-like and triangular columns and the dualtriangle-chevron-like capture chamber unit is about 210 μm to about 700μm in length and about 210 μm to about 700 μm in width.
 18. Themicrofluidic device according to claim 13, wherein the capture unitscomprising the pair of triangles and chevron-like chambers are organizedin rows or arranged randomly.
 19. The microfluidic device according toclaim 1, wherein the elliptical capture chambers have dimensions ofabout 100 μm to about 250 μm, and from 50 μm to 150 μm for major andminor axes and height respectively.
 20. The microfluidic deviceaccording to claim 1, wherein the capture agent is immobilized tocapture surfaces via a cross-linker N-[γ-maleimidobutyryloxy]succinimideester (GMBS).
 21. A method for detecting circulating tumor cells invitro, said method comprising (a) combining a test sample from a patientsuspected of having circulating tumor cells, and a non-lytic adenoviralsystem, and culture media for the cells, said adenoviral systemcomprising: (i) a first replication-defective adenoviral particle havingan adenoviral capsid in which an expression cassette is packaged, saidexpression cassette comprising an adenoviral 5′ ITR, an tumor-specificpromoter which is specifically activated in the presence of circulatingtumor cells, and an adenoviral 3′ ITR, wherein said adenoviral panicleis rendered replication defective and non-lytic by a deletion in one ormore adenoviral early genes; and (ii) a coding sequence for a reporterprotein which is expressed in the presence of circulating tumor cells,and an adenoviral 3′ ITR; (b) incubating the test sample and thenon-lytic adenoviral system for a sufficient time to permit expressionof the reporter protein, optionally replenishing the media; and (c)measuring reporter protein expression in the test samples and optionallyrepeating (c), whereby presence of reporter expression indicates thepresence of circulating tumor cells in the sample.
 22. The methodaccording to claim 21, wherein the promoter is selected from the groupconsisting of a human telomerase reverse transcriptase (hTERT), a humantelomerase RNA (hTR), a hTERC promoter, and a progression elevated genepromoter (PEG-Prom). 23-29. (canceled)
 30. The method according to claim21, wherein the first adenoviral particle (i) further comprisessequences encoding an endonuclease and (ii) further comprises anexpression cassette for the reporter gene expression cassette whichfurther comprises an endonuclease recognition site located both 5′ and3′ to the coding sequence and expression control sequences for thereporter gene. 31-36. (canceled)
 37. The method according to claim 21,wherein the reporter protein is selected from the group consisting of agreen fluorescent protein, enhanced green fluorescent protein, mCherry,red fluorescent protein, red fluorescent protein-turbo.
 38. The methodaccording to claim 21, further comprising enriching a blood sample fromthe patient prior to combining it with the non-lytic adenoviral system.39. The method according to claim 21, wherein the blood sample isenriched by: obtaining a whole blood sample from a patient; combiningthe whole blood sample with neutral buffered saline in an OncoQick tubehaving a conical bottom; centrifuging the tube for about 10 minutes toabout 60 minutes at about 500 relative centrifugal force (RCF) units toabout 3000 RCF units; harvesting the CTC-enriched layer to obtain aCTC-enriched pellet suspension; incubating the pellet suspension in awater bath; introducing cell culture media to allow pellet suspension inpreparation for the adenoviral system.
 40. The method according to claim39, wherein the whole blood sample is about 1 mL to about 15 mL. 41-42.(canceled)
 43. The method according to claim 40, wherein the whole bloodsample is combined with an equal volume of the buffered saline.
 44. Themethod according to claim 40, wherein the whole blood and bufferedsaline are chilled to about 0° C. to about 4° C. prior to centifruging.45. The method according to claim 40, wherein centrifuging is performedat about 20 minutes at about 1500 RCF units.
 46. The method according toclaim 40, wherein following collection of the CTC-enriched pelletsuspension, the remaining solution is extracted and placed in a separateconical tube and the first conical is washed to collect any residualCTCs.
 47. A product comprising a novel vector system for use in atelomerase based assay for detecting circulating tumor cells in vitro,said system comprising: (a) a first replication-defective adenoviralparticle having an adeno viral capsid in which an endonucleaseexpression cassette is packaged, said expression cassette comprising anadenoviral 5′ TTR, an hTERT promoter sequence which is activated in thepresence of circulating tumor cells, an endonuclease coding sequenceunder the direction of sequences which direct expression thereof, and anadenoviral 3′ ITR, wherein said adenoviral particle is renderedreplication defective by a deletion in one or more adenoviral earlygenes; and (b) a DNA molecule comprising an expression cassette for areporter gene comprising a coding sequence for the reporter protein andsequences which direct expression thereof, said expression cassetteflanked at its 5′ and 3′ ends by recognition sites for the endonuclease,and an adenoviral 3′ ITR.
 48. The product according to claim 47, whereinthe adenoviral capsid of (a) is human adenovirus
 5. 49. The productaccording to claim 47, wherein the marker expression cassette (b) is ona second replication-defective adenoviral panicle.
 50. (canceled) 51.The product according to claim 47, wherein the marker expressioncassette (b) is carried by the first replication-defective adenoviralparticle. 52-55. (canceled)